FcGammaRIIB Specific Antibodies and Methods of Use Thereof

ABSTRACT

The present invention relates to antibodies or fragments thereof that specifically bind the extracellular domain of FcγRIIB, particularly human FcγRIIB, and block the Fc binding site of human FcγRIIB. The invention provides methods of treating cancer and/or regulating immune complex-mediated cell activation by administering the antibodies of the invention to enhance an immune response. The invention also provides methods of breaking tolerance to an antigen by administering an antigen-antibody complex and an antibody of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/305,787, filed Dec. 15, 2005, which claims priority to U.S.Provisional Patent Application Ser. No. 60/636,663, filed Dec. 15, 2004.This application is also a continuation-in-part of U.S. patentapplication Ser. No. 10/524,134 filed on Feb. 11, 2005, which is aNational Stage Application under 35 U.S.C. §371 of PCT ApplicationSerial No. PCT/US03/25399, filed on Aug. 14, 2003, which claims priorityto U.S. Provisional Application Ser. No. 60/403,266, filed on Aug. 14,2002. This application is also a continuation-in-part of U.S. patentapplication Ser. No. 10/643,857 filed on Aug. 14, 2003, which claimspriority to U.S. Provisional Application Ser. No. 60/403,266, filed onAug. 14, 2002. This application is also a continuation-in-part of U.S.patent application Ser. No. 11/108,135, filed on Apr. 15, 2005, whichclaims priority to U.S. Provisional Application Ser. Nos. 60/562,804,60/582,044, 60/582,045 and 60/654,713, filed on Apr. 14, 2004, Jun. 21,2004, Jun. 21, 2004 and Feb. 18, 2005 respectively. All of theabove-identified applications are incorporated herein by reference intheir entireties.

FIELD OF THE INVENTION

The present invention relates to antibodies or fragments thereof thatspecifically bind the extracellular domain of FcγRIIB, particularlyhuman FcγRIIB, and block the Fc binding site of human FcγRIIB. Theinvention provides methods of treating cancer and/or regulating immunecomplex-mediated cell activation by administering the antibodies of theinvention to enhance an immune response. The invention also providesmethods of breaking tolerance to an antigen by administering anantigen-antibody complex and an antibody of the invention.

BACKGROUND OF THE INVENTION I. Fc Receptors and their Roles in theImmune System

The interaction of antibody-antigen complexes with cells of the immunesystem results in a wide array of responses, ranging from effectorfunctions such as antibody-dependent cytotoxicity, mast celldegranulation, and phagocytosis to immunomodulatory signals such asregulating lymphocyte proliferation and antibody secretion. All theseinteractions are initiated through the binding of the Fc domain ofantibodies or immune complexes to specialized cell surface receptors onhematopoietic cells. The diversity of cellular responses triggered byantibodies and immune complexes results from the structuralheterogeneity of Fc receptors. Fc receptors share structurally relatedligand binding domains which presumably mediate intracellular signaling.

The Fc receptors, members of the immunoglobulin gene superfamily ofproteins, are surface glycoproteins that can bind the Fc portion ofimmunoglobulin molecules. Each member of the family recognizesimmunoglobulins of one or more isotypes through a recognition domain onthe A chain of the Fc receptor. Fc receptors are defined by theirspecificity for immunoglobulin subtypes. Fc receptors for IgG arereferred to as FcγR, for IgE as FcεR, and for IgA as FcαR. Differentaccessory cells bear Fc receptors for antibodies of different isotype,and the isotype of the antibody determines which accessory cells will beengaged in a given response (reviewed by Ravetch J. V. et al. (1991) “FcReceptors,” Annu. Rev. Immunol. 9: 457-92; Gerber et al. (2001)“Stimulatory And Inhibitory Signals Originating From The MacrophageFcgamma Receptors,” Microbes and Infection, 3: 131-139; Billadeau et al.(2002), “ITAMs Versus ITIMs: Striking A Balance During Cell Regulation,”The Journal of Clinical Investigation, 2(109): 161-168; Ravetch J. V. etal. (2000) “Immune Inhibitory Receptors,” Science, 290: 84-89; Ravetchet al. (2001) “IgG Fc Receptors,” Annu. Rev. Immunol. 19:275-290;Ravetch (1994) “Fc Receptors: Rubor Redux,” Cell, 78(4): 553-560). Thedifferent Fc receptors, the cells that express them, and their isotypespecificity is summarized in Table 1 (adapted from IMMUNOBIOLOGY: THEIMMUNE SYSTEM IN HEALTH AND DISEASE, 4th ed. 1999, Elsevier ScienceLtd/Garland Publishing, New York).

A. Fcγ Receptors

Each member of this family is an integral membrane glycoprotein,possessing extracellular domains related to a C2-set ofimmunoglobulin-related domains, a single membrane spanning domain and anintracytoplasmic domain of variable length. There are three known FcγRs,designated FcγRI(CD64), FcγRII(CD32), and FcγRIII(CD16). The threereceptors are encoded by distinct genes; however, the extensive homologybetween the three family members suggest they arose from a commonprogenitor perhaps by gene duplication. This invention specificallyfocuses on FcγRII(CD32).

B. FcγRII(CD32)

FcγRII proteins are 40 KDa integral membrane glycoproteins which bindonly the complexed IgG due to a low affinity for monomeric Ig (10⁶ M⁻¹).This receptor is the most widely expressed FcγR, present on allhematopoietic cells, including monocytes, macrophages, B cells, NKcells, neutrophils, mast cells, and platelets. FcγRII has only twoimmunoglobulin-like regions in its immunoglobulin binding chain andhence a much lower affinity for IgG than FcγRI. There are three humanFcγRII genes (FcγRII-A, FcγRII-B, FcγRII-C), all of which bind IgG inaggregates or immune complexes.

Distinct differences within the cytoplasmic domains of FcγRII-A andFcγRII-B create two functionally heterogenous responses to receptorligation. The fundamental difference is that the A isoform initiatesintracellular signaling leading to cell activation such as phagocytosisand respiratory burst, whereas the B isoform initiates inhibitorysignals, e.g., inhibiting B-cell activation.

C. Signaling Through FcγRs

Both activating and inhibitory signals are transduced through the FcγRsfollowing ligation. These diametrically opposing functions result fromstructural differences among the different receptor isoforms. Twodistinct domains within the cytoplasmic signaling domains of thereceptor called Immunoreceptor Tyrosine based Activation Motifs (ITAMs)or Immunoreceptor Tyrosine based Inhibitory Motifs (ITIMS) account forthe different responses. The recruitment of different cytoplasmicenzymes to these structures dictates the outcome of the FcγR-mediatedcellular responses. ITAM-containing FcγR complexes include FcγRI,FcγRIIA, FcγRIIIA, whereas ITIM-containing complexes only includeFcγRIIB.

Human neutrophils express the FcγRIIA gene. FcγRIIA clustering viaimmune complexes or specific antibody cross-linking serves to aggregateITAMs along with receptor-associated kinases, which facilitate ITAMphosphorylation. ITAM phosphorylation serves as a docking site for Sykkinase, activation of which results in activation of downstreamsubstrates (e.g., PI₃K). Cellular activation leads to release ofproinflammatory mediators. The FcγRIIB gene is expressed on Blymphocytes; its extracellular domain is 96% identical to FcγRIIA andbinds IgG complexes in an indistinguishable manner. The presence of anITIM in the cytoplasmic domain of FcγRIIB defines this inhibitorysubclass of FcγR. Recently the molecular basis of this inhibition wasestablished. When colligated along with an activating FcγR, the ITIM inFcγRIIB becomes phosphorylated and attracts the SH2 domain of theinosital polyphosphate 5′-phosphatase (SHIP), which hydrolyzesphosphoinositol messengers that are released as a consequence ofITAM-containing FcγR-mediated tyrosine kinase activation, consequentlypreventing the influx of intracellular Ca⁺⁺. Thus, crosslinking ofFcγRIIB dampens the activating response to FcγR ligation and inhibitscellular responsiveness. B cell activation, B cell proliferation andantibody secretion is thus aborted.

TABLE 1 Receptors for the Fc Regions of Immunoglobulin Isotypes Effectof Receptor Binding Cell Type Ligation FcγRI IgG1 Macrophages,Neutrophils, Uptake (CD64 10⁸ M⁻¹ Eosinophils, Dendritic cellsStimulation Activation of respiratory burst Induction of killingFcγRII-A IgG1 Macrophages, Neutrophils, Uptake (CD32) 2 × 10⁶ M⁻¹Eosinophils, Dendritic Granule cells, Platelets, release Langerhan cellsFcγRII-B2 IgG1 Macrophages, Neutrophils, Uptake Inhibition (CD32) 2 ×10⁶ M⁻¹ Eosinophils of Stimulation FcγRII-BI IgG1 B cells, Mast cells Nouptake (CD32) 2 × 10⁶ M⁻¹ Inhibition of Stimulation FcγRIII IgG1 NKcells, Eosinophil Induction of (CD16) 5 × 10⁵ M⁻¹ macrophages,Neutrophils, Killing Mast Cells FcεRI IgG1 Mast cells, EosinophilSecretion of 10¹⁰ M⁻¹ Basophils granules FcαRI IgG1, IgA2 Macrophages,Neutropils Uptake Induction (CD89) 10⁷ M⁻¹ Eosinophils of killing

II. Diseases of Relevance

A. Cancer

A neoplasm, or tumor, is a neoplastic mass resulting from abnormaluncontrolled cell growth which can be benign or malignant. Benign tumorsgenerally remain localized. Malignant tumors are collectively termedcancers. The term “malignant” generally means that the tumor can invadeand destroy neighboring body structures and spread to distant sites tocause death (for review, see Robbins and Angell, 1976, Basic Pathology,2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-122). Cancer can arisein many sites of the body and behave differently depending upon itsorigin. Cancerous cells destroy the part of the body in which theyoriginate and then spread to other part(s) of the body where they startnew growth and cause more destruction.

More than 1.2 million Americans develop cancer each year. Cancer is thesecond leading case of death in the United States and if current trendscontinue, cancer is expected to be the leading cause of the death by theyear 2010. Lung and prostate cancer are the top cancer killers for menin the United States. Lung and breast cancer are the top cancer killersfor women in the United States. One in two men in the United States willbe diagnosed with cancer at some time during his lifetime. One in threewomen in the United States will be diagnosed with cancer at some timeduring her lifetime.

A cure for cancer has yet to be found. Current treatment options, suchas surgery, chemotherapy and radiation treatment, are oftentimes eitherineffective or present serious side effects.

Currently, cancer therapy may involve surgery, chemotherapy, hormonaltherapy and/or radiation treatment to eradicate neoplastic cells in apatient (See, for example, Stockdale, 1998, “Principles of CancerPatient Management”, in Scientific American: Medicine, vol. 3,Rubenstein and Federman, eds., Chapter 12, Section IV). Recently, cancertherapy could also involve biological therapy or immunotherapy. All ofthese approaches pose significant drawbacks for the patient. Surgery,for example, may be contraindicated due to the health of the patient ormay be unacceptable to the patient. Additionally, surgery may notcompletely remove the neoplastic tissue. Radiation therapy is onlyeffective when the neoplastic tissue exhibits a higher sensitivity toradiation than normal tissue, and radiation therapy can also oftenelicit serious side effects. Hormonal therapy is rarely given as asingle agent and although can be effective, is often used to prevent ordelay recurrence of cancer after other treatments have removed themajority of the cancer cells. Biological therapies/immunotherapies arelimited in number and may produce side effects such as rashes orswellings, flu-like symptoms, including fever, chills and fatigue,digestive tract problems or allergic reactions.

With respect to chemotherapy, there are a variety of chemotherapeuticagents available for treatment of cancer. A significant majority ofcancer chemotherapeutics act by inhibiting DNA synthesis, eitherdirectly or indirectly by inhibiting the biosynthesis of thedeoxyribonucleotide triphosphate precursors, to prevent DNA replicationand concomitant cell division (See, for example, Gilman et al., Goodmanand Gilman's: The Pharmacological Basis of Therapeutics, Eighth Ed.(Pergamom Press, New York, 1990)). These agents, which includealkylating agents, such as nitrosourea, anti-metabolites, such asmethotrexate and hydroxyurea, and other agents, such as etoposides,campathecins, bleomycin, doxorubicin, daunorubicin, etc., although notnecessarily cell cycle specific, kill cells during S phase because oftheir effect on DNA replication. Other agents, specifically colchicineand the vinca alkaloids, such as vinblastine and vincristine, interferewith microtubule assembly resulting in mitotic arrest. Chemotherapyprotocols generally involve administration of a combination ofchemotherapeutic agents to increase the efficacy of treatment.

Despite the availability of a variety of chemotherapeutic agents,chemotherapy has many drawbacks (See, for example, Stockdale, 1998,“Principles Of Cancer Patient Management” in Scientific AmericanMedicine, vol. 3, Rubenstein and Federman, eds., ch. 12, sect. 10).Almost all chemotherapeutic agents are toxic, and chemotherapy causessignificant, and often dangerous, side effects, including severe nausea,bone marrow depression, immunosuppression, etc. Additionally, even withadministration of combinations of chemotherapeutic agents, many tumorcells are resistant or develop resistance to the chemotherapeuticagents. In fact, those cells resistant to the particularchemotherapeutic agents used in the treatment protocol often prove to beresistant to other drugs, even those agents that act by mechanismsdifferent from the mechanisms of action of the drugs used in thespecific treatment; this phenomenon is termed pleiotropic drug ormultidrug resistance. Thus, because of drug resistance, many cancersprove refractory to standard chemotherapeutic treatment protocols.

There is a significant need for alternative cancer treatments,particularly for treatment of cancer that has proved refractory tostandard cancer treatments, such as surgery, radiation therapy,chemotherapy, and hormonal therapy. A promising alternative isimmunotherapy, in which cancer cells are specifically targeted by cancerantigen-specific antibodies. Major efforts have been directed atharnessing the specificity of the immune response, for example,hybridoma technology has enabled the development of tumor selectivemonoclonal antibodies (See Green et al. (2000) “Monoclonal AntibodyTherapy For Solid Tumors,” Cancer Treat Rev., 26: 269-286; Weiner L M(1999) “Monoclonal Antibody Therapy Of Cancer,” Semin Oncol. 26(suppl.14):43-51), and in the past few years, the Food and Drug Administrationhas approved the first MAbs for cancer therapy: RITUXAN (rituximab)(anti-CD20) for non-Hodgkin's Lymphoma and HERCEPTIN® (trastuzumab)[anti-(c-erb-2/HER-2)] for metastatic breast cancer (S. A. Eccles (2001)“Monoclonal Antibodies Targeting Cancer: ‘Magic Bullets’ Or Just TheTrigger?,” Breast Cancer Res., 3: 86-90). However, the potency ofantibody effector function, e.g., to mediate antibody dependent cellularcytotoxicity (“ADCC”) is an obstacle to such treatment. Methods toimprove the efficacy of such immunotherapy are thus needed.

B. Allergy

Immune-mediated allergic (hypersensitivity) reactions are classifiedinto four types (1-IV) according to the underlying mechanisms leading tothe expression of the allergic symptoms. Type I allergic reactions arecharacterized by IgE-mediated release of vasoactive substances such ashistamine from mast cells and basophils. The release of these substancesand the subsequent manifestation of allergic symptoms are initiated bythe cross-linking of allergen-bound IgE to its receptor on the surfaceof mast cells and basophils. In individuals suffering from type Iallergic reactions, exposure to an allergen for a second time leads tothe production of high levels of IgE antibodies specific for theallergen as a result of the involvement of memory B and T cells in the3-cell interaction required for IgE production. The high levels of IgEantibodies produced cause an increase in the cross-linking of IgEreceptors on mast cells and basophils by allergen-bound IgE, which inturn leads to the activation of these cells and the release of thepharmacological mediators that are responsible for the clinicalmanifestations of type I allergic diseases.

Two receptors with differing affinities for IgE have been identified andcharacterized. The high affinity receptor (FcεRI) is expressed on thesurface of mast cells and basophils. The low affinity receptor(FcεRII/CD23) is expressed on many cell types including B cells, Tcells, macrophages, eosinophils and Langerhan cells. The high affinityIgE receptor consists of three subunits (alpha, beta and gamma chains).Several studies demonstrate that only the alpha chain is involved in thebinding of IgE, whereas the beta and gamma chains (which are eithertransmembrane or cytoplasmic proteins) are required for signaltransduction events. The identification of IgE structures required forIgE to bind to the FcεRI on mast cells and basophils is of utmostimportance in devising strategies for treatment or prevention ofIgE-mediated allergies. For example, the elucidation of the IgEreceptor-binding site could lead to the identification of peptides orsmall molecules that block the binding of IgE to receptor-bearing cellsin vivo.

Currently, IgE-mediated allergic reactions are treated with drugs suchas antihistamines and corticosteroids which attempt to alleviate thesymptoms associated with allergic reactions by counteracting the effectsof the vasoactive substances released from mast cells and basophils.High doses of antihistamines and corticosteroids have deleterious sideeffects (e.g., central nervous system disturbance, constipation, etc).Thus, other methods for treating type I allergic reactions are needed.

One approach to the treatment of type I allergic disorders has been theproduction of monoclonal antibodies which react with soluble (free) IgEin serum, block IgE from binding to its receptor on mast cells andbasophils, and do not bind to receptor-bound IgE (i.e., they arenon-anaphylactogenic). Two such monoclonal antibodies are in advancedstages of clinical development for treatment of IgE-mediated allergicreactions (see, e.g., Chang, T. W. (2000) “The Pharmacological Basis OfAnti-IgE Therapy,” Nature Biotechnology 18:157-162).

One of the most promising treatments for IgE-mediated allergic reactionsis the active immunization against appropriate non-anaphylactogenicepitopes on endogenous IgE. Stanworth et al. (U.S. Pat. No. 5,601,821)described a strategy involving the use of a peptide derived from theCεH4 domain of the human IgE coupled to a heterologous carrier proteinas an allergy vaccine. However, this peptide has been shown not toinduce the production of antibodies that react with native soluble IgE.Further, Hellman (U.S. Pat. No. 5,653,980) proposed anti-IgE vaccinecompositions based on fusion of full length CεH2-CεH3 domains(approximately 220 amino acid long) to a foreign carrier protein.However, the antibodies induced by the anti-IgE vaccine compositionsproposed in Hellman will most likely it result in anaphylaxis sinceantibodies against some portions of the CεH2 and CεH3 domains of the IgEmolecule have been shown to cross-link the IgE receptor on the surfaceof mast cell and basophils and lead to production of mediators ofanaphylaxis (See, e.g., Stadler et al. (1993) “Biological Activities OfAnti-IgE Antibodies,” Int. Arch. Allergy and Immunology 102:121-126).Therefore, a need remains for treatment of IgE-mediated allergicreactions which do not induce anaphylactic antibodies.

The significant concern over induction of anaphylaxis has resulted inthe development of another approach to the treatment of type I allergicdisorders consisting of mimotopes that could induce the production ofanti-IgE polyclonal antibodies when administered to animals (See, e.g.,Rudolf, et al. (1998) “Epitope-Specific Antibody Response To IgE ByMimotope Immunization,” Journal of Immunology 160:3315-3321). Kricek etal. (International Publication No. WO 97/31948) screened phage-displayedpeptide libraries with the monoclonal antibody BSWI7 to identify peptidemimotopes that could mimic the conformation of the IgE receptor binding.These mimotopes could presumably be used to induce polyclonal antibodiesthat react with free native IgE, but not with receptor-bound IgE as wellas block IgE from binding to its receptor. Kriek et al. disclosedpeptide mimotopes that are not homologous to any part of the IgEmolecule and are thus different from peptides disclosed in the presentinvention.

As evidenced by a survey of the art, there remains a need for enhancingthe therapeutic efficacy of current methods of treating or preventingdisorders such as cancer or allergy. In particular, there is a need forenhancing the effector function, particularly, the cytotoxic effect oftherapeutic antibodies used in treatment of cancer. The current state ofthe art is also lacking in treating or preventing allergy disorders(e.g., either by antibody therapy or vaccine therapy).

SUMMARY OF THE INVENTION

The extracellular domains of FcγRIIA and FcγRIIB are 95% identical andthus they share numerous epitopes. However, FcγRIIA and FcγRIIB exhibitvery different activities. The fundamental difference is that theFcγRIIA initiates intracellular signaling leading to cell activationsuch as phagocytosis and respiratory burst, whereas the FcγRIIBinitiates inhibitory signaling. Prior to this invention, to theknowledge of the inventors, antibodies known to distinguish among nativehuman FcγRIIA and native human FcγRIIB have not been identified; in viewof their distinctive activities and role in modulating immune responses,such antibodies that recognize native FcγRIIB, and not native FcγRIIA,are needed. The present invention is based, in part, on the discovery ofsuch FcγRIIB-specific antibodies. As used herein, “native FcγRIIB orFcγRIIA” means FcγRIIB or FcγRIIA which is endogenously expressed in acell and is present on the cell surface of that cell or recombinantlyexpressed in a mammalian cell and present on the cell surface, but isnot FcγRIIB or FcγRIIA expressed in a bacterial cell or denatured,isolated FcγRIIB or FcγRIIA.

The invention relates to an antibody or a fragment thereof thatspecifically binds FcγRIIB, particularly human FcγRIIB, moreparticularly native human FcγRIIB, and blocks the Fc binding domain ofFcγRIIB, particularly human FcγRIIB, more particularly native humanFcγRIIB. Preferably, the antibodies of the invention bind theextracellular domain of native human FcγRIIB. In certain embodiments ofthe invention, the antibody or a fragment thereof binds FcγRIIB with atleast 2 times greater affinity than said antibody or a fragment thereofbinds FcγRIIA. In other embodiments of the invention, the antibody or afragment thereof, binds FcγRIIB with at least 4 times, at least 6 times,at least 8 times, at least 10 times, at least 100 times, at least 1000times, at least 10⁴, at least 10⁵, at least 10⁶, at least 10⁷, or atleast 10⁸ times greater affinity than said antibody or a fragmentthereof binds FcγRIIA In a preferred embodiment, said antibody or afragment thereof binds FcγRIIB with 100 times, 1000 times, 10⁴ times,10⁵ times, 10⁶ times, 10⁷ times, or 10⁸ times greater affinity than saidantibody or a fragment thereof binds FcγRIIA. Preferably, these bindingaffinities are determined with the monomeric IgG, and not the aggregatedIgG, and binding is via the variable domain (e.g., Fab fragments of theantibodies have binding characteristic similar to the full immunolobulinmolecule).

In one particular embodiment, the anti-FcγRIIB antibodies block theligand binding site of FcγRIIB. In a further specific embodiment, theblocking activity can block the negative regulation ofimmune-complex-triggered activation and consequently enhance the immuneresponse. In a further specific embodiment, the enhanced immune responseis an increase in antibody-dependent cellular response. In anotherspecific embodiment, the anti-FcγRIIB antibodies of the invention blockcrosslinking of FcγRIIB receptors to B cell and/or Fc receptors, leadingto B cell, mast cell, dendritic cell, or macrophage activation.

In a preferred embodiment, the antibody or fragment thereof blockscrosslinking of FcγRIIB to an immunoreceptor tyrosine-based activationmotif (ITAM) containing activating receptor, preferably enhancing theactivity of an activating receptor. ITAM-containing receptors, includeFc receptors, and BCR-associated Igα. In certain embodiments, theblocking leads to B cell, mast cell, dendritic cell, or macrophageactivation.

In certain embodiments, the Fc receptor is a FcεR or a FcγR, preferablyFcεRI. Preferably, an an FcεRI dependent activity is modulated, forexample, modulation of calcium mobilization and/or modulation ofdegranulation.

In one embodiment, the FcγRIIB-specific antibody in accordance with theinvention is not the monoclonal antibody designated KB61, as disclosedin Pulford et al. (1986) “A New Monoclonal Antibody (KB61) Recognizing ANovel Antigen Which Is Selectively Expressed On A Subpopulation Of HumanB Lymphocytes,” Immunology 57:71-76 or the monoclonal antibody II8D2disclosed in Weinrich et al. (1996) “Epitope Mapping Of New MonoclonalAntibodies Recognizing Distinct Human FcRII (CD32) Isoforms,” Hybridoma15: 109-116. In a specific embodiment, the FcγRIIB-specific antibody ofthe invention does not bind to the same epitope and/or does not competefor binding with the monoclonal antibody KB61 or the monoclonal antibodyMAbII8D2. Preferably, the antibody of the invention does not bind theamino sequence SDPNFSI (SEQ ID NO:59), corresponding to positions 176 to182 of the FcγRIIB 2 isoform (SEQ ID NO:60).

SEQ ID NO: 60: MGILSFLPVL ATESDWADCK SPQPWGHMLL WTAVLFLAPV AGTPAPPKAV 50 LKLEPQWINV LQEDSVTLTC RGTHSPESDS IQWFHNGNLI PTHTQPSYRF 100KANNNDSGEY TCQTGQTSLS DPVHLTVLSE WLVLQTPHLE FQEGETIVLR 150 CHSWKDKPLVKVTFFQNGKS KKFSRSDPNF SIPQANHSHS GDYHCTGNIG 200 YTLYSSKPVT ITVQAPSSSPMGIIVAVVTG IAVAAIVAAV VALIYCRKKR 250 ISANPTNPDE ADKVGAENTI TYSLLMHPDALEEPDDQNRI 290

In a particular embodiment, the invention relates to an isolatedantibody or a fragment thereof that specifically binds FcγRIIB with agreater affinity than said antibody or a fragment thereof binds FcγRIIA,and the constant domain of said antibody further has an enhancedaffinity for at least one or more Fc activation receptors. In yetanother specific embodiment, said Fc activation receptor is FcγRIII.

In one embodiment of the invention said antibody or a fragment thereofblocks the IgG binding site of FcγRIIB and blocks the binding ofaggregated labeled IgGs to FcγRIIB in, for example, a blocking ELISAassay. In one particular embodiment, said antibody or a fragment thereofblocks the binding of aggregated labeled IgGs in an ELISA blocking assayby at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 99.9%. In yet anotherparticular embodiment, the antibody or a fragment thereof completelyblocks the binding of said aggregated labeled IgG in said ELISA assay.

In another embodiment of the invention, said antibody or a fragmentthereof blocks the IgG binding site of FcγRIIB and blocks the binding ofaggregated labeled IgG to FcγRIIB, as determined by a double-stainingFACS assay.

The invention encompasses the use of antibodies that modulate (i.e.,agonize or antagonize) the activity of FcγRIIB. In one embodiment of theinvention, the antibodies of the invention agonize at least one activityof FcγRIIB, i.e., elicit signaling. Although not intending to be boundby any mechanism of action, agonistic antibodies of the invention maymimic clustering of FcγRIIB leading to dampening of the activatingresponse to FcγR ligation and inhibition of cellular responsiveness.

In another embodiment of the invention, the antibodies of the inventionantagonize at least one activity of FcγRIIB, i.e., block signaling. Forexample, the antibodies of the invention block the binding of aggregatedIgGs to FcγRIIB.

The invention provides antibodies that inhibit FcεRI-induced mast cellactivation. The invention further provides anti-FcγRIIB antibodies thatinhibit FcγRIIA-mediated macrophage activation in monocytic cells. Theinvention also provides anti-FcγRIIB antibodies that inhibit B-cellreceptor mediated signaling.

In certain embodiments, the Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that the modifiedFc region has an altered binding affinity to an Fc receptor. Preferably,the antibody or fragment thereof has an increased binding affinity toFcγRIIB or FcγRIII. Preferred amino acid modifications comprise asubstitution at position 265 or 297. More preferably, the amino acidmodification is a substitution at position 265 with alanine or asubstitution at position 297 with glutamine.

In a preferred embodiment, the invention provides a monoclonal antibodyproduced by clone 2B6 or 3H7, having ATCC accession numbers PTA-4591 andPTA-4592, respectively. In another embodiment, the invention provides anisolated antibody, or a fragment thereof, that competes for binding withthe monoclonal antibody produced by clone 2B6 or 3H7 and binds FcγRIIB,preferably native human FcγRIIB with a greater affinity than saidantibody or a fragment thereof binds FcγRIIA, preferably native humanFcγRIIA and/or binds to the same epitope of FcγRIIB as the monoclonalantibody produced from clone 2B6 or 3H7 and binds FcγRIIB with a greateraffinity than said antibody or a fragment thereof binds FcγRIIA.Furthermore, the invention provides hybridoma cell line 2B6 or 3H7,having ATCC accession numbers PTA-4591 and PTA-4592, respectively.

The methods of the invention also encompass polynucleotides that encodethe antibodies of the invention. In one embodiment, the inventionprovides an isolated nucleic acid sequence encoding a heavy chain or alight chain of an antibody or a fragment thereof that specifically bindsFcγRIIB with greater affinity than said antibody or a fragment thereofbinds FcγRIIA. In another embodiment, the invention provides an isolatednucleic acid sequence encoding a heavy chain or a light chain of anantibody or a fragment thereof that specifically binds FcγRIIB andblocks the Fc binding domain of FcγRIIB. The invention also relates to avector comprising said nucleic acid. The invention further provides avector comprising a first nucleic acid molecule encoding a heavy chainand a second nucleic acid molecule encoding a light chain, said heavychain and light chain being of an antibody or a fragment thereof thatspecifically binds FcγRIIB with greater affinity than said antibody or afragment thereof binds FcγRIIA. The invention further provides a vectorcomprising a first nucleic acid molecule encoding a heavy chain and asecond nucleic acid molecule encoding a light chain, said heavy chainand light chain being of an antibody or a fragment thereof thatspecifically binds FcγRIIB and blocks the Fc binding domain of FcγRIIB.In one specific embodiment, said vector is an expression vector. Theinvention further provides host cells containing the vectors of orpolynucleotides encoding the antibodies of the invention. Preferably,the invention encompasses polynucleotides encoding heavy and lightchains of the antibodies produced by the deposited hybridoma clones,having ATCC accession numbers PTA-4591 and PTA-4592, respectively, orportions thereof, e.g., CDRs, variable domains, humanized versionsthereof, etc.

The invention further provides methods for the production of antibodiesof the invention or fragments thereof. The antibodies of the inventionor fragments thereof can be produced by any method known in the art forthe production of antibodies, in particular, by secretion from culturedhybridoma cells, chemical synthesis or by recombinant expressiontechniques known in the art. In one specific embodiment, the inventionrelates to a method for recombinantly producing a FcγRIIB-specificantibody, said method comprising:

-   -   (i.) culturing under conditions suitable for the expression of        said antibody in a medium, a host cell containing a first        nucleic acid molecule, operably linked to a heterologous        promoter and a second nucleic acid operably linked to the same        or a different heterologous promoter, said first nucleic acid        and second nucleic acid encoding a heavy chain and a light        chain, respectively, of an antibody or a fragment thereof that        specifically binds FcγRIIB with greater affinity than said        antibody or a fragment thereof binds FcγRIIA or an antibody or a        fragment thereof that specifically binds FcγRIIB and blocks the        Fc binding domain of FcγRIIB; and    -   (ii.) recovery of said antibody from said medium.

Preferably, the antibodies of the invention are monoclonal antibodies,and more preferably, humanized or human antibodies. In certainembodiments, an antibody fragment of the invention is a F(ab′)₂ fragmentor F(ab) fragment. In one specific preferred embodiment, the antibodiesof the invention bind to the extracellular domain of human FcγRIIB,particularly native human FcγRIIB. In another specific embodiment, theantibodies of the invention specifically or selectively recognize one ormore epitopes of FcγRIIB, particularly native human FcγRIIB. Anotherembodiment of the invention encompasses the use of phage displaytechnology to increase the affinity of the antibodies of the inventionfor FcγRIIB. Any screening method known in the art can be used toidentify mutant antibodies with increased avidity for FcγRIIB (e.g.,ELISA). In another specific embodiment, antibodies of the invention arescreened using antibody screening assays well known in the art (e.g.,BIACORE assays) to identify antibodies with K_(off) rate less than3×10⁻³ s⁻¹.

Activating and inhibitory Fc receptors, e.g., FcγRIIA and FcγRIIB, arecritical for the balanced function of these receptors and propercellular immune responses. The invention encompasses the use of theantibodies of the invention for the treatment of any disease related toloss of such balance and regulated control in the Fc receptor signalingpathway. Thus, the FcγRIIB antibodies of the invention have uses inregulating the immune response. The FcγRIIB antibodies of the inventioncan also be used to alter certain effector functions to enhance, forexample, therapeutic antibody-mediated cytotoxicity.

The antibodies of the invention are useful for prevention or treatmentof cancer, for example, in one embodiment, as a single agent therapy. Inone embodiment of the invention, the antibodies of the invention areuseful for prevention or treatment of B-cell malignancies, particularlynon-Hodgkin's lymphoma or chronic lymphocytic leukemia. In a preferredembodiment, the antibodies of the invention are used for the treatmentand/or prevention of melanoma. In another embodiment, the antibodies areuseful for prevention or treatment of cancer, particularly inpotentiating the cytotoxic activity of cancer antigen-specifictherapeutic antibodies with cytotoxic activity to enhance tumor cellkilling and/or enhancing antibody dependent cytotoxic cellular (“ADCC”)activity, complement dependent cytotoxic (“CDC”) activity, orphagocytosis of the therapeutic antibodies. The invention provides amethod of treating cancer in a patient having a cancer characterized bya cancer antigen, said method comprising administering to said patient atherapeutically effective amount of a first antibody or a fragmentthereof that specifically binds FcγRIIB with greater affinity than saidantibody or a fragment thereof binds FcγRIIA, and a second antibody thatspecifically binds said cancer antigen and is cytotoxic. The inventionalso provides a method of treating cancer in a patient having a cancercharacterized by a cancer antigen, said method comprising administeringto said patient a therapeutically effective amount of an antibody or afragment thereof that specifically binds FcγRIIB, particularly nativehuman FcγRIIB with greater affinity than said antibody or a fragmentthereof binds FcγRIIA, preferably native human FcγRIIA, and the constantdomain of which further has an increased affinity for one or more Fcactivation receptors, when the antibody is monomeric, such as FcγRIIIA,and an antibody that specifically binds said cancer antigen and iscytotoxic. In one particular embodiment, said Fc activation receptor isFcγRIIIA.

The invention also provides a method of treating cancer in a patienthaving a cancer characterized by a cancer antigen, said methodcomprising administering to said patient a therapeutically effectiveamount of an antibody or a fragment thereof that specifically binds saidcancer antigen and a therapeutically effective amount of an antibody orfragment thereof that specifically binds the extracellular domain ofhuman FcγRIIB and blocks the Fc binding site of human FcγRIIB.

In another embodiment, the invention provides a method of enhancing anantibody mediated cytotoxic effect in a subject being treated with acytotoxic antibody, said method comprising administering to said patientan antibody of the invention or a fragment thereof, in an amountsufficient to enhance the cytotoxic effect of said cytotoxic antibody.In yet another embodiment, the invention provides a method of enhancingan antibody-mediated cytotoxic effect in a subject being treated with acytotoxic antibody, said method comprising administering to said patientan antibody of the invention or a fragment thereof, further having anenhanced affinity for an Fc activation receptor, when monomeric, in anamount sufficient to enhance the cytotoxic effect of said cytotoxicantibody. In yet another embodiment, the invention provides a methodfurther comprising the administration of one or more additional cancertherapies.

In yet another embodiment, the invention provides a method of regulatingimmune-complex mediated cell activation in a patient, said methodcomprising administering to said patient a therapeutically effectiveamount of an antibody or fragment thereof that specifically binds theextracellular domain of human FcγRIIB and blocks the Fc binding site ofhuman FcγRIIB. In a preferred embodiment, administration of the antibodyor fragment thereof results in an enhanced immune response, such as anincrease in an antibody-dependent cellular response. In anotherpreferred embodiment, the immune complex mediated cell activation is Bcell activation, mast cell activation, dendritic cell activation ormacrophage activation.

In another embodiment, the invention provides a method of breakingtolerance to an antigen in a patient, said method comprisingadministering to a patient in need thereof:

-   -   (A.) an antigen-antibody complex comprising said antigen; and    -   (B.) an antibody or fragment thereof that specifically binds the        extracellular domain of human FcγRIIB and blocks the Fc binding        site of human FcγRIIB, thereby breaking tolerance in said        patient to said antigen.

The antibody or fragment thereof can be administered before,concurrently with, or after administration of said antigen-antibodycomplex.

The invention further provides a pharmaceutical composition comprising:

-   -   (i.) a therapeutically effective amount of an antibody or        fragment thereof that specifically binds the extracellular        domain of human FcγRIIB and blocks the Fc binding site of human        FcγRIIB;    -   (ii.) a cytotoxic antibody that specifically binds a cancer        antigen; and    -   (iii.) a pharmaceutically acceptable carrier.

In a preferred embodiment, the antibody or fragment thereof is a humanor humanized antibody. In another preferred embodiment, the antibody orfragment thereof that specifically binds the extracellular domain ofhuman FcγRIIB and blocks the Fc binding site of human FcγRIIB blockscrosslinking of FcγRIIB to a Fc receptor. In yet another preferredembodiment, the antibody or fragment thereof that specifically binds theextracellular domain of human FcγRIIB and blocks the Fc binding site ofhuman FcγRIIB comprises a Fc region comprising at least one amino acidmodification relative to a wild-type Fc region, such that the modifiedFc region has an altered binding affinity to a Fc receptor. In apreferred embodiment, the amino acid modification comprises asubstitution at position 265 or 297, preferably a substitution atposition 265 with alanine or a substitution at position 297 withglutamine. In certain embodiments, the cytotoxic antibody is HERCEPTIN®(trastuzumab), RITUXAN® rituximab, IC14, PANOREX® (edrecolomab),IMC-225, VITAXIN™, CAMPATH® (alemtuzumab) 1H/LDP-03, LYMPHOCIDE®(epratuzumab), or ZEVLIN® (ibritumomab tiuxetan).

The invention encompasses the use of the antibodies of the invention incombination with any therapeutic antibody that mediates its therapeuticeffect through cell killing to potentiate the antibody's therapeuticactivity. In one particular embodiment, the antibodies of the inventionpotentiate the antibody's therapeutic activity by enhancingantibody-mediated effector function. In another embodiment of theinvention, the antibodies of the invention potentiate the cytotoxicantibody's therapeutic activity by enhancing phagocytosis andopsonization of the targeted tumor cells. In yet another embodiment ofthe invention, the antibodies of the invention potentiate thetherapeutic activity of the antibody by enhancing antibody-dependentcell-mediated cytotoxicity (“ADCC”) in destruction of the targeted tumorcells.

In some embodiments, the invention encompasses use of the antibodies ofthe invention in combination with a therapeutic antibody that does notmediate its therapeutic effect through cell killing to potentiate theantibody's therapeutic activity. In a specific embodiment, the inventionencompasses use of the antibodies of the invention in combination with atherapeutic apoptosis inducing antibody with agonistic activity, e.g.,anti-Fas antibody. Therapeutic apoptosis inducing antibodies may bespecific for any death receptor known in the art for the modulation ofapoptotic pathway, e.g., TNFR receptor family member.

The invention encompasses using the antibodies of the invention to blockmacrophage mediated tumor cell progression and metastasis. Theantibodies of the invention are particularly useful in the treatment ofsolid tumors, where macrophage infiltration occurs. The antagonisticantibodies of the invention are particularly useful for controlling,e.g., reducing or eliminating, tumor cell metastasis, by reducing oreliminating the population of macrophages that are localized at thetumor site. The invention further encompasses antibodies thateffectively deplete or eliminate immune effector cells other thanmacrophages that express FcγRIIB, e.g., dendritic cells. Effectivedepletion or elimination of immune effector cells using the antibodiesof the invention may range from a reduction in population of theeffector cells by 50%, 60%, 70%, 80%, preferably 90%, and mostpreferably 99%.

In some embodiments, the agonistic antibodies of the invention areparticularly useful for the treatment of tumors of non-hematopoieticorigin, including tumors of melanoma cells.

I. DEFINITIONS

As used herein, the term “specifically binds to FcγRIIB” and analogousterms refer to antibodies or fragments thereof that specifically bind toFcγRIIB or a fragment thereof and do not specifically bind to other Fcreceptors, in particular to FcγRIIA. Further, it is understood to oneskilled in the art that an antibody that specifically binds to FcγRIIB,may bind through the variable domain or the constant domain of theantibody. If the antibody that specifically binds to FcγRIIB bindsthrough its variable domain, it is understood to one skilled in the artthat it is not aggregated, i.e., is monomeric. An antibody thatspecifically binds to FcγRIIB may bind to other peptides or polypeptideswith lower affinity as determined by, e.g., immunoassays, BIAcore, orother assays known in the art. Preferably, antibodies or fragments thatspecifically bind to FcγRIIB or a fragment thereof do not cross-reactwith other antigens. Antibodies or fragments that specifically bind toFcγRIIB can be identified, for example, by immunoassays, BIAcore, orother techniques known to those of skill in the art. An antibody or afragment thereof binds specifically to FcγRIIB when it binds to FcγRIIBwith higher affinity than to any cross-reactive antigen as determinedusing experimental techniques, such as western blots, radioimmunoassays(RIA) and enzyme-linked immunosorbent assays (ELISAs). (See, e.g., Paul,ed., 1989, Fundamental Immunology Second Edition, Raven Press, New Yorkat pages 332-336 for a discussion regarding antibody specificity.)

As used herein, the term “native FcγRIIB” refers to FcγRIIB which isendogenously expressed and present on the surface of a cell. In someembodiments, “native FcγRIIB” encompasses a protein that isrecombinantly expressed in a mammalian cell. Preferably, the nativeFcγRIIB is not expressed in a bacterial cell, i.e., E. coli. Mostpreferably, the native FcγRIIB is not denatured, i.e., it is in itsbiologically active conformation.

As used herein, the term “native FcγRIIA” refers to FcγRIIA which isendogenously expressed and present on the surface of a cell. In someembodiments, “native FcγRIIA” encompasses a protein that isrecombinantly expressed in a mammalian cell. Preferably, the nativeFcγRIIA is not expressed in a bacterial cell, i.e., E. coli. Mostpreferably the native FcγRIIA is not denatured, i.e., it is in itsbiologically active conformation.

As used herein, the terms “antibody” and “antibodies” refer tomonoclonal antibodies, multispecific antibodies, human antibodies,humanized antibodies, synthetic antibodies, chimeric antibodies,camelized antibodies, single-chain Fvs (scFv), single chain antibodies,Fab fragments, F(ab′) fragments, disulfide-linked Fvs (sdFv),intrabodies, and anti-idiotypic (anti-Id) antibodies (including, e.g.,anti-Id and anti-anti-Id antibodies to antibodies of the invention), andepitope-binding fragments of any of the above. In particular, antibodiesinclude immunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site. Immunoglobulin molecules can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁and IgA₂) or subclass.

As used herein, the term “derivative” in the context of polypeptides orproteins refers to a polypeptide or protein that comprises an amino acidsequence that has been altered by the introduction of amino acid residuesubstitutions, deletions or additions. The term “derivative” as usedherein also refers to a polypeptide or protein that has been modified,i.e, by the covalent attachment of any type of molecule to thepolypeptide or protein. For example, but not by way of limitation, anantibody may be modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. A derivative polypeptide or protein may beproduced by chemical modifications using techniques known to those ofskill in the art, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Further, a derivative polypeptide or protein derivative possesses asimilar or identical function as the polypeptide or protein from whichit was derived.

The term “derivative” as used herein refers to a polypeptide thatcomprises an amino acid sequence of a FcγRIIB polypeptide, a fragment ofa FcγRIIB polypeptide, an antibody that immunospecifically binds to aFcγRIIB polypeptide, or an antibody fragment that immunospecificallybinds to a FcγRIIB polypeptide, that has been altered by theintroduction of amino acid residue substitutions, deletions or additions(i.e., mutations). In some embodiments, an antibody derivative orfragment thereof comprises amino acid residue substitutions, deletionsor additions in one or more CDRs. The antibody derivative may havesubstantially the same binding, better binding, or worse binding whencompared to a non-derivative antibody. In specific embodiments, one,two, three, four, or five amino acid residues of the CDR have beensubstituted, deleted or added (i.e., mutated). The term “derivative” asused herein also refers to a FcγRIIB polypeptide, a fragment of aFcγRIIB polypeptide, an antibody that immunospecifically binds to aFcγRIIB polypeptide, or an antibody fragment that immunospecificallybinds to a FcγRIIB polypeptide which has been modified, i.e., by thecovalent attachment of any type of molecule to the polypeptide. Forexample, but not by way of limitation, a FcγRIIB polypeptide, a fragmentof a FcγRIIB polypeptide, an antibody, or antibody fragment may bemodified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. A derivative of an FcγRIIB polypeptide, a fragment of aFcγRIIB polypeptide, an antibody, or antibody fragment may be modifiedby chemical modifications using techniques known to those of skill inthe art, including, but not limited to, specific chemical cleavage,acetylation, formulation, metabolic synthesis of tunicamycin, etc.Further, a derivative of a FcγRIIB polypeptide, a fragment of a FcγRIIBpolypeptide, an antibody, or antibody fragment may contain one or morenon-classical amino acids. In one embodiment, a polypeptide derivativepossesses a similar or identical function as a FcγRIIB polypeptide, afragment of a FcγRIIB polypeptide, an antibody, or antibody fragmentdescribed herein. In another embodiment, a derivative of a FcγRIIBpolypeptide, a fragment of a FcγRIIB polypeptide, an antibody, orantibody fragment has an altered activity when compared to an unalteredpolypeptide. For example, a derivative antibody or fragment thereof canbind to its epitope more tightly or be more resistant to proteolysis.

As used herein, the terms “disorder” and “disease” are usedinterchangeably to refer to a condition in a subject. In particular, theterm “autoimmune disease” is used interchangeably with the term“autoimmune disorder” to refer to a condition in a subject characterizedby cellular, tissue and/or organ injury caused by an immunologicreaction of the subject to its own cells, tissues and/or organs. Theterm “inflammatory disease” is used interchangeably with the term“inflammatory disorder” to refer to a condition in a subjectcharacterized by inflammation, preferably chronic inflammation.Autoimmune disorders may or may not be associated with inflammation.Moreover, inflammation may or may not be caused by an autoimmunedisorder. Thus, certain disorders may be characterized as bothautoimmune and inflammatory disorders.

As used herein, the term “cancer” refers to a neoplasm or tumorresulting from abnormal uncontrolled growth of cells. As used herein,cancer explicitly includes, leukemias and lymphomas. The term “cancer”refers to a disease involving cells that have the potential tometastasize to distal sites and exhibit phenotypic traits that differfrom those of non-cancer cells, for example, formation of colonies in athree-dimensional substrate such as soft agar or the formation oftubular networks or weblike matrices in a three-dimensional basementmembrane or extracellular matrix preparation. Non-cancer cells do notform colonies in soft agar and form distinct sphere-like structures inthree-dimensional basement membrane or extracellular matrixpreparations. Cancer cells acquire a characteristic set of functionalcapabilities during their development, albeit through variousmechanisms. Such capabilities include evading apoptosis,self-sufficiency in growth signals, insensitivity to anti-growthsignals, tissue invasion/metastasis, limitless explicative potential,and sustained angiogenesis. The term “cancer cell” is meant to encompassboth pre-malignant and malignant cancer cells. In some embodiments,cancer refers to a benign tumor, which has remained localized. In otherembodiments, cancer refers to a malignant tumor, which has invaded anddestroyed neighboring body structures and spread to distant sites. Inyet other embodiments, the cancer is associated with a specific cancerantigen.

As used herein, the term “immunomodulatory agent” and variations thereofincluding, but not limited to, immunomodulatory agents, refer to anagent that modulates a host's immune system. In certain embodiments, animmunomodulatory agent is an immunosuppressant agent. In certain otherembodiments, an immunomodulatory agent is an immunostimulatory agent.Immunomodatory agents include, but are not limited to, small molecules,peptides, polypeptides, fusion proteins, antibodies, inorganicmolecules, mimetic agents, and organic molecules.

As used herein, the term “epitope” refers to a fragment of a polypeptideor protein having antigenic or immunogenic activity in an animal,preferably in a mammal, and most preferably in a human. An epitopehaving immunogenic activity is a fragment of a polypeptide or proteinthat elicits an antibody response in an animal. An epitope havingantigenic activity is a fragment of a polypeptide or protein to which anantibody immunospecifically binds as determined by any method well-knownto one of skill in the art, for example by immunoassays. Antigenicepitopes need not necessarily be immunogenic.

As used herein, the term “fragment” refers to a peptide or polypeptidecomprising an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least contiguous 80 amino acid residues, atleast contiguous 90 amino acid residues, at least contiguous 100 aminoacid residues, at least contiguous 125 amino acid residues, at least 150contiguous amino acid residues, at least contiguous 175 amino acidresidues, at least contiguous 200 amino acid residues, or at leastcontiguous 250 amino acid residues of the amino acid sequence of anotherpolypeptide. In a specific embodiment, a fragment of a polypeptideretains at least one function of the polypeptide. Preferably, antibodyfragments are epitope binding fragments.

As used herein, the term “humanized antibody” refers to forms ofnon-human (e.g., murine) antibodies that are chimeric antibodies whichcontain minimal sequence derived from non-human immunoglobulin. For themost part, humanized antibodies are human immunoglobulins (recipientantibody) in which hypervariable region residues of the recipient arereplaced by hypervariable region residues from a non-human species(donor antibody) such as mouse, rat, rabbit or non-human primate havingthe desired specificity, affinity, and capacity. In some instances,Framework Region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiesmay comprise residues which are not found in the recipient antibody orin the donor antibody. These modifications are made to further refineantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FRs are those of a human immunoglobulinsequence. The humanized antibody optionally also will comprise at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin that immunospecifically binds to a FcγRIIBpolypeptide, that has been altered by the introduction of amino acidresidue substitutions, deletions or additions (i.e., mutations). In someembodiments, a humanized antibody is a derivative. Such a humanizedantibody comprises amino acid residue substitutions, deletions oradditions in one or more non-human CDRs. The humanized antibodyderivative may have substantially the same binding, better binding, orworse binding when compared to a non-derivative humanized antibody. Inspecific embodiments, one, two, three, four, or five amino acid residuesof the CDR have been substituted, deleted or added (i.e., mutated). Forfurther details in humanizing antibodies, see European Patent Nos. EP239,400, EP 592,106, and EP 519,596; International Publication Nos. WO91/09967 and WO 93/17105; U.S. Pat. Nos. 5,225,539, 5,530,101,5,565,332, 5,585,089, 5,766,886, and 6,407,213; and Padlan (1991) “APossible Procedure For Reducing The Immunogenicity Of Antibody VariableDomains While Preserving Their Ligand-Binding Properties,” MolecularImmunology 28(4/5):489-498; Studnicka et al. (1994) “Human-EngineeredMonoclonal Antibodies Retain Full Specific Binding Activity ByPreserving Non-Cdr Complementarity-Modulating Residues,” ProteinEngineering 7:805-814; and Roguska et al. (1994) “Humanization Of MurineMonoclonal Antibodies Through Variable Domain Resurfacing,” Proc. Nat.Acad. Sci. 91:969-973; Tan et al. (2002) “‘Superhumanized’ Antibodies:Reduction Of Immunogenic Potential By Complementarity-Determining RegionGrafting With Human Germline Sequences: Application To An Anti-CD28,” J.Immunol. 169:1119 1125; Caldas et al. (2000) “Design And Synthesis OfGermline-Based Hemi-Humanized Single-Chain Fv Against The CD18 SurfaceAntigen,” Protein Eng. 13:353 360; Morea et al. (2000) “AntibodyModeling: Implications For Engineering And Design,” Methods 20:267 279;Baca et al. (1997) “Antibody Humanization Using Monovalent PhageDisplay,” J. Biol. Chem. 272:10678-10684; Roguska et al. (1996) “AComparison Of Two Murine Monoclonal Antibodies Humanized By CDR-GraftingAnd Variable Domain Resurfacing,” Protein Eng. 9:895 904; Couto et al.(1995) “Designing Human Consensus Antibodies With Minimal PositionalTemplates,” Cancer Res. 55 (23 Supp):5973s 5977s; Couto et al. (1995)“Anti-BA46Monoclonal Antibody Mc3: Humanization Using A Novel PositionalConsensus And In Vivo And In Vitro Characterization,” Cancer Res.55:1717 22; Sandhu (1994) “A Rapid Procedure For The Humanization OfMonoclonal Antibodies,” Gene 150:409 410; Pedersen et al. (1994)“Comparison Of Surface Accessible Residues In Human And MurineImmunoglobulin Fv Domains. Implication For Humanization Of MurineAntibodies,” J. Mol. Biol. 235:959 973; Jones et al. (1986) “ReplacingThe Complementarity-Determining Regions In A Human Antibody With ThoseFrom A Mouse,” Nature 321:522-525; Riechmann et al. (1988) “ReshapingHuman Antibodies For Therapy,” Nature 332:323-327; and Presta (1992)“Antibody Engineering,” Curr. Op. Biotech. 3:394-398.

As used herein, the term “hypervariable region” refers to the amino acidresidues of an antibody that are responsible for antigen binding. Thehypervariable region comprises amino acid residues from a“Complementarity Determining Region” or “CDR” (i.e., residues 24-34(L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variabledomain; Kabat et al. Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md. (1991)) and/or those residues from a “hypervariable loop” (i.e.,residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavychain variable domain; Chothia et al. (1987) “Canonical structures forthe hypervariable regions of immunoglobulins,” J. Mol. Biol.196:901-917). CDR residues for Eph099B-208.261 and Eph099B-233.152 arelisted in Table 1 of WO/2003/094859. “Framework Region” or “FR” residuesare those variable domain residues other than the hypervariable regionresidues as herein defined.

As used herein, the terms “single-chain Fv” or “scFv” refer to antibodyfragments comprise the VH and VL domains of antibody, wherein thesedomains are present in a single polypeptide chain. Generally, the Fvpolypeptide further comprises a polypeptide linker between the VH and VLdomains that enables the scFv to form the desired structure for antigenbinding. For a review of sFv, see Pluckthun in THE PHARMACOLOGY OFMONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269-315 (1994). In specific embodiments,scFvs include bi-specific scFvs and humanized scFvs.

As used herein, the terms “nucleic acids” and “nucleotide sequences”include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g.,mRNA), combinations of DNA and RNA molecules or hybrid DNA/RNAmolecules, and analogs of DNA or RNA molecules. Such analogs can begenerated using, for example, nucleotide analogs, which include, but arenot limited to, inosine or tritylated bases. Such analogs can alsocomprise DNA or RNA molecules comprising modified backbones that lendbeneficial attributes to the molecules such as, for example, nucleaseresistance or an increased ability to cross cellular membranes. Thenucleic acids or nucleotide sequences can be single-stranded,double-stranded, may contain both single-stranded and double-strandedportions, and may contain triple-stranded portions, but preferably isdouble-stranded DNA.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, a subject is preferably a mammal suchas a non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) and aprimate (e.g., monkey and human), most preferably a human.

As used herein, the terms “treat,” “treating” and “treatment” refer tothe eradication, reduction or amelioration of symptoms of a disease ordisorder related to the loss of regulation in the Fc receptor signalingpathway or to enhance the therapeutic efficacy of another therapy, e.g.,a therapeutic antibody, vaccine therapy. In some embodiments, treatmentrefers to the eradication, removal, modification, or control of primary,regional, or metastatic cancer tissue that results from theadministration of one or more therapeutic agents. In certainembodiments, such terms refer to the minimizing or delaying the spreadof cancer resulting from the administration of one or more therapeuticagents to a subject with such a disease.

As used herein, the phrase “side effects” encompasses unwanted andadverse effects of a prophylactic or therapeutic agent. Adverse effectsare always unwanted, but unwanted effects are not necessarily adverse.An adverse effect from a prophylactic or therapeutic agent might beharmful or uncomfortable or risky. Side effects from chemotherapyinclude, but are not limited to, gastrointestinal toxicity such as, butnot limited to, early and late-forming diarrhea and flatulence, nausea,vomiting, anorexia, leukopenia, anemia, neutropenia, asthenia, abdominalcramping, fever, pain, loss of body weight, dehydration, alopecia,dyspnea, insomnia, dizziness, mucositis, xerostomia, and kidney failure,as well as constipation, nerve and muscle effects, temporary orpermanent damage to kidneys and bladder, flu-like symptoms, fluidretention, and temporary or permanent infertility. Side effects fromradiation therapy include but are not limited to fatigue, dry mouth, andloss of appetite. Side effects from biological therapies/immunotherapiesinclude but are not limited to rashes or swellings at the site ofadministration, flu-like symptoms such as fever, chills and fatigue,digestive tract problems and allergic reactions. Side effects fromhormonal therapies include but are not limited to nausea, fertilityproblems, depression, loss of appetite, eye problems, headache, andweight fluctuation. Additional undesired effects typically experiencedby patients are numerous and known in the art, see, e.g., thePHYSICIANS' DESK REFERENCE (56^(th) ed., 2002), which is incorporatedherein by reference in its entirety.

As used herein, a “therapeutically effective amount” refers to thatamount of the therapeutic agent sufficient to treat or manage a diseaseor disorder associated with FcγRIIB and any disease related to the lossof regulation in the Fc receptor signaling pathway or to enhance thetherapeutic efficacy of another therapy, e.g., therapeutic antibody,vaccine therapy, etc. A therapeutically effective amount may refer tothe amount of therapeutic agent sufficient to delay or minimize theonset of disease, e.g., delay or minimize the spread of cancer. Atherapeutically effective amount may also refer to the amount of thetherapeutic agent that provides a therapeutic benefit in the treatmentor management of a disease. Further, a therapeutically effective amountwith respect to a therapeutic agent of the invention means that amountof therapeutic agent alone, or in combination with other therapies, thatprovides a therapeutic benefit in the treatment or management of adisease, e.g., sufficient to enhance the therapeutic efficacy of atherapeutic antibody sufficient to treat or manage a disease. Used inconnection with an amount of FcγRIIB antibody of the invention, the termcan encompass an amount that improves overall therapy, reduces or avoidsunwanted effects, or enhances the therapeutic efficacy of or synergieswith another therapeutic agent.

As used herein, the terms “prophylactic agent” and “prophylactic agents”refer to any agent(s) that can be used in the prevention of a disorder,or prevention of recurrence or spread of a disorder. A prophylacticallyeffective amount may refer to the amount of prophylactic agentsufficient to prevent the recurrence or spread of hyperproliferativedisease, particularly cancer, or the occurrence of such in a patient,including but not limited to those predisposed to hyperproliferativedisease, for example those genetically predisposed to cancer orpreviously exposed to carcinogens. A prophylactically effective amountmay also refer to the amount of the prophylactic agent that provides aprophylactic benefit in the prevention of disease. Further, aprophylactically effective amount with respect to a prophylactic agentof the invention means that amount of prophylactic agent alone, or incombination with other agents, that provides a prophylactic benefit inthe prevention of disease. Used in connection with an amount of anFcγRIIB antibody of the invention, the term can encompass an amount thatimproves overall prophylaxis or enhances the prophylactic efficacy of orsynergies with another prophylactic agent, such as but not limited to atherapeutic antibody. In certain embodiments, the term “prophylacticagent” refers to an agonistic FcγRIIB-specific antibody. In otherembodiments, the term “prophylactic agent” refers to an antagonisticFcγRIIB-specific antibody. In certain other embodiments, the term“prophylactic agent” refers to cancer chemotherapeutics, radiationtherapy, hormonal therapy, biological therapy (e.g., immunotherapy),and/or FcγRIIB antibodies of the invention. In other embodiments, morethan one prophylactic agent may be administered in combination.

As used herein, the terms “manage,” “managing” and “management” refer tothe beneficial effects that a subject derives from administration of aprophylactic or therapeutic agent, which does not result in a cure ofthe disease. In certain embodiments, a subject is administered one ormore prophylactic or therapeutic agents to “manage” a disease so as toprevent the progression or worsening of the disease.

As used herein, the terms “prevent”, “preventing” and “prevention” referto the prevention of the recurrence or onset of one or more symptoms ofa disorder in a subject resulting from the administration of aprophylactic or therapeutic agent.

As used herein, the term “in combination” refers to the use of more thanone prophylactic and/or therapeutic agents. The use of the term “incombination” does not restrict the order in which prophylactic and/ortherapeutic agents are administered to a subject with a disorder, e.g.,hyperproliferative cell disorder, especially cancer. A firstprophylactic or therapeutic agent can be administered prior to (e.g., 1minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours,4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeksbefore), concomitantly with, or subsequent to (e.g., 1 minute, 5minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after)the administration of a second prophylactic or therapeutic agent to asubject which had, has, or is susceptible to a disorder. Theprophylactic or therapeutic agents are administered to a subject in asequence and within a time interval such that the agent of the inventioncan act together with the other agent to provide an increased benefitthan if they were administered otherwise. Any additional prophylactic ortherapeutic agent can be administered in any order with the otheradditional prophylactic or therapeutic agents.

II. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B: Direct binding of the antibody produced from the 3H7clone to FcγRIIB and FcγRIIA.

FIGS. 1A-1B: The direct binding of antibodies from some of the hybridomacultures to the FcγRIIs were compared to a commercially availableanti-FcγRII antibody in an ELISA assay where the plate was coated withthe receptors. Different dilutions (1:10) of the supernatants wereincubated on the plate. The bound antibodies were detected with a goatanti-mouse HRP conjugated antibody and the absorbance was monitored at650 nm.

FIGS. 1C-1D: The direct binding of the antibody from the 3H7 hybridomaculture (supernatant n. 7 from the FIGS. 1A-B), in crude (FIG. 1C) andpurified form (FIG. 1D), to FcγRIIA and FcγRIIB, were compared using thesame ELISA assay as in 1A.

FIG. 2: Competition in binding to FcγRIIB of the antibody produced fromthe 3H7 hybridoma and aggregated biotinylated human IgG.

The ability of the 3H7 antibody to compete with aggregated biotinylatedhuman IgG for binding to FcγRIIB was measured using a blocking ELISAexperiment. The ELISA plate coated with FcγRIIB was incubated with thesupernatant containing the 3H7 antibody and with a supernatant from thesame hybridoma cells but not containing antibody (negative control).Different dilutions (1:3) starting from 200 ng/well, of aggregatedbiotinylated human IgG were then added to the plate and the boundaggregates were detected with Streptavidin-Horse-Radish Peroxidaseconjugated, the reaction was developed with TMB and the absorbance wasmonitored at 650 nm.

FIG. 3: Comparison of the direct binding of the 3H7 antibody to FcγRIIBproduced in a bacterial or in a mammalian system.

Direct binding of the 3H7 antibody to FcγRIIB was measured using anELISA assay. Binding to the bacterial or mammalian produced FcγRIIB wascompared. The antibody titration started from the straight supernatantfollowed by 1:10 dilutions. The bound antibody was detected with a goatanti-mouse HRP conjugated antibody, the reaction was developed with TMBand the absorbance was monitored at 650 nm.

FIG. 4: Direct binding of the 3H7 antibody to FcγRIIA, FcγRIIB andFcγRIIIA.

The direct binding of the purified 3H7 antibody to FcγRIIA, FcγRIIB andFcγRIIIA expressed in a mammalian system were compared using the ELISAassay. ELISA plate was coated with the three receptors (100 ng/well).Different dilutions of the purified 3H7 antibody were incubated on thecoated plate. A goat anti-mouse-HRP conjugated antibody was used fordetection of the bound specific antibody, the reaction was developedwith TMB and the absorbance was monitored at 650 nm.

FIGS. 5A-5C: Comparison of the direct binding ability to FcγRIIA andFcγRIIB of the antibody purified from clone 2B6 compared to other threecommercially available monoclonal antibodies against FcγRII.

The binding of 2B6 antibody to FcγRIIA (FIG. 5B) and FcγRIIB (FIG. 5A)is compared to that of three other commercially available antibodiesraised against FcγRII. The ELISA format used is the same described inFIG. 4.

FIG. 5C shows IIB/IIA binding of 2B6 and FL18.26.

FIGS. 6A and 6B: Competition in binding of the antibody produced fromclone 2B6 and aggregated biotinylated human IgG to FcγRIIB.

FIG. 6A: The ability of the antibody present in the supernatant from theclone 2B6 to compete for binding to FcγRIIB with aggregated biotinylatedhuman IgG was measured using a blocking ELISA experiment. The 2B6antibody competition ability was compared to that of a negativesupernatant from hybridoma and to that of 3H7 antibody. An ELISA platecoated with FcγRIIB was incubated with different dilutions (1:10) of thesupernatants. After washes the plate was incubated with a fixed amountof aggregated biotinylated human IgG (1 mg/well) and the boundaggregates were detected with Streptavidin-HRP conjugated. The reactionwas developed with TMB and the absorbance was monitored at 650 nm.

FIG. 6B: The same blocking ELISA described in panel A was performed withpurified 2B6 antibody and the data from one concentration of blockingantibody used (4 mg/well) were represented in a bar diagram. The 2B6ability to block aggregated human IgG binding to FcγRIIB was compared tothat of a mouse IgG1 isotype control.

FIGS. 7A-7C: Competition of 2B6 antibody and aggregated biotinylatedhuman IgG in binding to FcγRIIB using a double-staining FACS assay.

A double staining FACS assay was performed to characterize the 2B6antibody using CHO-K1 cells that had been stably transfected withfull-length mammalian FcγRIIB.

FIG. 7A: The transfectant cells were stained with mouse IgG1 isotypecontrol followed by a goat anti-mouse-FITC conjugated antibody andStreptavidin-PE.

FIG. 7B: The transfectant cells were stained with aggregatedbiotinylated human IgG after being stained with mouse IgG1 isotypecontrol and labeled with a goat anti-mouse-FITC conjugated antibody todetect the bound monoclonal antibody and with Streptavidin-PE conjugatedto detect the bound aggregates.

FIG. 7C: The cells were stained with 2B6 antibody, the antibody wasremoved by washes and the cells were incubated with aggregatedbiotinylated human IgG. Cells were washed and labeled with a goatanti-mouse-FITC conjugated antibody to detect the bound monoclonalantibody and with Streptavidin-PE conjugated to detect the boundaggregates.

FIGS. 8A-8C: Biacore analysis of 2B6 and KB6.1 antibody binding.

Binding of 2B6 antibody and KB6.1 antibody to surface linked CD32B (FIG.8A), CD32A (H131) (FIG. 8B), and CD32A (R131) (FIG. 8C) were compared.

FIGS. 9A-9C: Monoclonal anti FcγRIIB antibodies and CD20 co-stain ofhuman B lymphocytes.

Cells from human blood (“buffy coat”) were stained with anti-CD20-FITCconjugated antibody, to select the B lymphocytes population, as well as3H7 and 2B6. The bound anti-FcγRIIB antibodies were detected with a goatanti-mouse-PE conjugated antibody.

FIG. 9A: Cells were co-stained with anti-CD20-FITC antibody and mouseIgG1 isotype control.

FIG. 9B: Cells were co-stained with anti-CD20-FITC antibody and 3H7antibody.

FIG. 9C: Cells were co-stained with anti-CD20-FITC antibody and 2B6antibody.

FIGS. 10A and 10B: Staining of CHO cells expressing FcγRIIB.

FIGS. 10A-10B: CHO/IIB cells were stained with mouse IgG1 isotypecontrol (FIG. 10A) and 3H7 antibody (FIG. 10B).

FIGS. 10C-10D: CHO/IIB cells were stained with mouse IgG1 isotypecontrol (FIG. 10C) and 2B6 antibody (FIG. 10D).

FIGS. 11A-11G: Staining of CHO cells expressing FcγRIIB.

CHO cells expressing huFcγRIIB were incubated with the anti-CD32Bantibody 3H7 (FIG. 11A), 2B6 (FIG. 11B), 2E1 (FIG. 11C), 2H9 (FIG. 11D),1D5 (FIG. 11E), 2D11 (FIG. 11F) and 1F2 (FIG. 11G). Cells were washedand 9 μg/ml of aggregated human IgG were added to the cells on ice. Thehuman aggregated IgG were detected with goat anti-human-IgG FITCconjugated. Samples were analyzed by FACS . . . isotype control+goatanti huIgG-FITC, —isotype control+aggregated humanIgG+goat antihumanIgG-FITC, —anti-CD32B antibody+aggregated humanIgG+goat antihumanIgG-FITC. The amount of each antibody bound to the receptor on thecells was also detected (inset) on a separate set of samples using agoat anti-mouse PE conjugated antibody.

FIGS. 12A-12J: Flow cytometry analysis of CD32B expression intransformed cell lines using CD32B specific antibody, 2B6, and CD32A/Breactive antibody, FLI8.26.

Cell lines: transfected 293H cells expressing CD32A (FIG. 12A, 12B) orCD32B (FIGS. 12C, 12D), Burkitt's lymphoma cell lines, Daudi (FIGS. 12E,12F) and Raji (FIGS. 12G, 12H), and the monocytic cell line, THP-1(FIGS. 12I, 12J).

FIGS. 13A-13P: Staining of Human PBMCs with 2B6, 3H7 and IV.3Antibodies.

Human PBMCs were stained with 2B6 (FIGS. 13B,13C, 13H, 13K and 13L), 3H7(FIGS. 13D, 13E, 13I, 13M and 13N), and 1V.3 (FIGS. 13F, 13G, 13J, 13Oand 13P) antibodies, as indicated on the right side of the panel,followed by a goat anti-mouse-Cyanine(Cy5) conjugated antibody (twocolor staining using anti-CD20-FITC conjugated antibody for Blymphocytes (FIGS. 13B, 13D and 13F), anti-CD14-PE conjugated antibodyfor monocytes (FIGS. 13K, 13M and 13O), anti-CD56-PE conjugated antibodyfor NK cells (FIGS. 13H, 13I and 13J) and anti-CD16-PE conjugatedantibody for granulocytes (FIGS. 13C, 13E, 13G, 13L, 13N and 13P). FIG.13A demonstrates staining results for monocytes, B lymphocytes andgranulocytes.

FIGS. 14A and 14B: β-Hexaminidase Release Assay.

FIG. 14A: Schematic representation of β-hexaminidase release assay.Transfectants expressing human FcγRIIB were sensitized with mouse IgEand challenged with F(ab′)₂ fragments of a polyclonal goat anti-mouseIgG to aggregate FcγRI. Crosslinking occurs because of the ability ofthe polyclonal antibody to recognize the light chain of the murine IgEantibody bound to FcγRI. Transfectants sensitized with murine IgE andpreincubated with 2B6 antibody were also challenged with F(ab′)₂fragments of a polyclonal goat anti-mouse IgG to cross link FcγRI toFcγRIIB.

FIG. 14B: β-hexosaminidase release induced by goat anti-mouse F(ab)₂fragment (GAM F(ab)₂) in RBL-2H3 cells expressing huFcγRIIB. Cells werestimulated with various concentration of GAM F(ab)₂ (0.03 μg/ml to 30μg/ml) after sensitization with mouse IgE (0.01 μg/ml) and IgG1 or withpurified 2B6 antibody (3 μg/ml) panel. After 1 hour at 37° C., thesupernatant was collected and the cells were lysed. β-hexosaminidaseactivity released in the supernatant and within the cells was determinedby a colorimetric assay using p-nitrophenyl N-acetyl-β-D-glucosaminide.The released β-hexosaminidase activity was expressed as a percentage ofthe released activity relative to the total activity.

FIGS. 15A-15C: 2B6 is capable of functionally blocking the Fc bindingsite of CD32B and prevent co-ligation of activating and inhibitoryreceptors.

FIG. 15A: Schematic representation of the experimental model.

FIGS. 15B and 15C: RBL-2H3/CD32B cells were stimulated with BSA-DNP-FITCcomplex in the presence of human IgG1, with BSA-DNP-FITC complexed withchimeric D265A4-4-20 in the presence or not of 3 μg/ml of F(ab)2fragments of 2B6 (FIG. 15B). Cells were also stimulated withBSA-DNP-FITC complex in the presence of human IgG1, with BSA-DNP-FITCcomplexed with chimeric 4-4-20 in the presence or not of 3 μg/ml ofF(ab)₂ fragments of 2B6 (FIG. 15C). After 30 minutes, the supernatantwas collected and the cells were lysed. B-hexosaminidase activityreleased in the supernatant and within the cells was determined by acolorimetric assay using p-nitrophenyl N-acetyl-β-D-glucosaminide. Thereleased β-hexosaminidase activity was expressed as a percentage of thereleased activity relative to the total activity.

FIGS. 16A-16C: Ovarian and Breast carcinoma cell lines express Her2/neuto varying levels.

Staining of Ovarian IGROV-1 (FIG. 16A) with purified ch4D5, OvarianOVCAR-8 with purified 4D5 antibody (FIG. 16B), and Breast cancer cells(FIG. 16C) with purified ch4D5 followed by goat anti-human-conjugated tophycoerythrin (PE). The relevant isotype control IgG1 is indicated theleft of the staining with anti-Her2neu antibody.

FIGS. 17A-17C: Elutriated Monocytes express all FcγRs.

MDM obtained from donor 1, propagated in human serum (FIGS. 14A, 14C,14E and 14G) or human serum and GM-CSF (FIGS. 14B, 14D, 14F and 14H);MDM obtained from donor 2; propagated in human serum (FIGS. 14I, 14K,14M and 14O) or human serum and GM-CSF (FIGS. 14J, 14L, 14N and 14P);and Monocytes thawed and stained immediately (FIGS. 14Q-14T).Monocyte-derived macrophages were stained with anti-bodies specific forhuman FcγR receptor. The solid histogram in each plot represents thebackground staining. The clear histogram within each panel representsthe staining with specific anti-human FcγR antibodies.

FIGS. 18A and 18B: Ch4D5 mediates effective ADCC with ovarian and breastcancer cell lines using PBMC.

Specific lysis subtracted from antibody-independent lysis is shown forOvarian tumor cell line, IGROV-1 (FIG. 18A) at an effector:target ratioof 75:1, and for Breast tumor cell line SKBR-3 (FIG. 18B) at aneffector:target ratio of 50:1 with different concentration of ch4D5 asindicated.

FIGS. 19A-19C: Histochemical staining of human ovarian ascites showstumors cells and other inflammatory cells.

FIG. 19A: H & E stain on ascites of a patient with ovarian tumor. Threeneoplastic cells can be identified by the irregular size and shape,scattered cytoplasm, and irregular dense nuclei.

FIG. 19B: Giemsa stain of unprocessed ascites from a patient with seroustumor of the ovary shows two mesothelial cells placed back to backindicated by short arrows. Also shown is a cluster of five malignantepithelial cells indicated by the long arrow. Erythrocytes are visiblein the background.

FIG. 19C: Giemsa stain of another patient with serous tumor of the ovaryindicating a cluster of cells composed of mesothelial cells,lymphocytes, and epithelial neoplastic cells (arrow).

FIG. 20: In vitro ADCC assay of ch2B6 and aglycosylated ch2B6 in Daudicells.

ch2B6 antibody mediates in vitro ADCC in CD32B expressing daudi cells.

DESCRIPTION OF THE PREFERRED EMBODIMENTS I. FcγRIIB-Specific Antibodies

The invention encompasses antibodies (preferably monoclonal antibodies)or fragments thereof that specifically bind FcγRIIB, preferably humanFcγRIIB, more preferably native human FcγRIIB with a greater affinitythan said antibodies or fragments thereof bind FcγRIIA, preferably humanFcγRIIA, more preferably native human FcγRIIA. Preferably, theantibodies of the invention bind the extracellular domain of nativehuman FcγRIIB. In certain embodiments, the antibodies or fragmentsthereof bind to FcγRIIB with an affinity greater than two-fold, fourfold, 6 fold, 10 fold, 20 fold, 50 fold, 100 fold, 1000 fold, 10⁴ fold,10⁵ fold, 10⁶ fold, 10⁷ fold, or 10⁸ fold than said antibodies orfragments thereof bind FcγRIIA.

The invention also encompasses antibodies or fragments thereof thatspecifically bind FcγRIIB, particularly human FcγRIIB, more particularlynative human FcγRIIB, and blocks the Fc binding domain of FcγRIIB,particularly human FcγRIIB, more particularly native human FcγRIIB.Preferably, the antibodies of the invention bind the extracellulardomain of native human FcγRIIB. In certain embodiments, the antibody orfragment thereof blocks crosslinking of FcγRIIB to an immunoreceptortyrosine-based activation motif (ITAM) containing activating receptor.ITAM containing receptors include, but are not limited to Fc Receptors(CD64, CD32A, CD16, CD23, FcεRI, etc.); TCR-associated CD3γ, CD3δ, CD3ε,and ζ chains; BCR-associated Igα (CD79A) and Igβ (CD79B) chains; DAP12;as well as several virally encoded transmembrane molecules. SeeBilladeau et al. (2002), “ITAMs Versus ITIMs: Striking A Balance DuringCell Regulation,” The Journal of Clinical Investigation, 2(109):161-168; herein incorporated by reference in its entirety. In preferredembodiments, this blocking enhances the activity of the activatingreceptor and/or leads to B cell, mast cell, dendritic cell, ormacrophage activation. In certain embodiments, the Fc receptor is anFcεR or an FcγR, preferably FcεRI. In preferred embodiments, an FcεRIdependent activity is modulated. In more preferred embodiments, theFcεRI dependent activity is modulation of calcium mobilization and/ormodulation of degranulation.

In one particular embodiment, the antibody is a mouse monoclonalantibody produced by clone 2B6 or 3H7, having ATCC accession numbersPTA-4591 and PTA-4592, respectively. Hybridomas producing antibodies ofthe invention have been deposited with the American Type CultureCollection (10801 University Blvd., Manassas, Va. 20110-2209) on Aug.13, 2002 under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedures, and assigned accession numbers PTA-4591(for hybridoma producing 2B6) and PTA-4592 (for hybridoma producing3H7), respectively and are incorporated herein by reference.

In a preferred embodiment, the antibodies of the invention are human orhave been humanized, preferably a humanized version of the antibodyproduced by clone 3H7 or 2B6. In other preferred embodiments, theantibodies of the invention are human or have been humanized, preferablya humanized version of the antibody produced by clone 1D5, 2E1, 2H9,2D11, or 1F2. Humanized versions of FcγRIIB-specific antibodies aredescribed in U.S. application Ser. No. 11/126,978, filed May 10, 2005,herein incorporated by reference in its entirety.

In yet another preferred embodiment, the antibodies of the inventionfurther do not bind Fc activation receptors, e.g., FcγIIIA, FcγIIIB,etc. In one embodiment, the FcγRIIB-specific antibody in accordance withthe invention is not the monoclonal antibody designated KB61, asdisclosed in Pulford et al. (1986) “A New Monoclonal Antibody (KB61)Recognizing A Novel Antigen Which Is Selectively Expressed On ASubpopulation Of Human B Lymphocytes,” Immunology 57:71-76, or themonoclonal antibody designated MAbII8D2 as disclosed in Weinrich et al.(1996) “Epitope Mapping Of New Monoclonal Antibodies RecognizingDistinct Human FcRII (CD32) Isoforms,” Hybridoma 15: 109-116. In aspecific embodiment, the FcγRIIB-specific antibody of the invention doesnot bind to the same epitope and/or does not compete with binding withthe monoclonal antibody KB61 or II8D2. Preferably, the antibody of theinvention does not bind the amino sequence SDPNFSI (SEQ ID NO:59),corresponding to positions 176 to 182 of FcγRIIB 2 isoform (SEQ IDNO:60).

The invention also encompasses other antibodies, preferably monoclonalantibodies or fragments thereof that specifically bind FcγRIIB,preferably human FcγRIIB, more preferably native human FcγRIIB, producedby clones including but not limited to 1D5, 2E1, 2H9, 2D11, and 1F2having ATCC Accession numbers, PTA-5958, PTA-5961, PTA-5962, PTA-5960,PTA-5959, respectively. Hybridomas producing the above-identified cloneswere deposited with the American Type Culture Collection (10801University Blvd., Manassas, Va. 20110-2209) on May 7, 2004, respectivelyand are incorporated herein by reference.

In a particular embodiment, the antibodies of the invention, orfragments thereof, agonize at least one activity of FcγRIIB. In oneembodiment of the invention, said activity is inhibition of B cellreceptor-mediated signaling. In another embodiment, the agonisticantibodies of the invention inhibit activation of B cells, B cellproliferation, antibody production, intracellular calcium influx of Bcells, cell cycle progression, or activity of one or more downstreamsignaling molecules in the FcγRIIB signal transduction pathway. In yetanother embodiment, the agonistic antibodies of the invention enhancephosphorylation of FcγRIIB or SHIP recruitment. In a further embodimentof the invention, the agonistic antibodies inhibit MAP kinase activityor Akt recruitment in the B cell receptor-mediated signaling pathway. Inanother embodiment, the agonistic antibodies of the invention agonizeFcγRIIB-mediated inhibition of FcεRI signaling. In a particularembodiment, said antibodies inhibit FcεRI-induced mast cell activation,calcium mobilization, degranulation, cytokine production, or serotoninrelease. In another embodiment, the agonistic antibodies of theinvention stimulate phosphorylation of FcγRIIB, stimulate recruitment ofSHIP, stimulate SHIP phosphorylation and its association with Shc, orinhibit activation of MAP kinase family members (e.g., Erk1, Erk2, JNK,p38, etc.). In yet another embodiment, the agonistic antibodies of theinvention enhance tyrosine phosphorylation of p62dok and its associationwith SHIP and rasGAP. In another embodiment, the agonistic antibodies ofthe invention inhibit FcγR-mediated phagocytosis in monocytes ormacrophages.

In another embodiment, the antibodies of the invention, or fragmentsthereof antagonize at least one activity of FcγRIIB. In one embodiment,said activity is activation of B cell receptor-mediated signaling. In aparticular embodiment, the antagonistic antibodies of the inventionenhance B cell activity, B cell proliferation, antibody production,intracellular calcium influx, or activity of one or more downstreamsignaling molecules in the FcγRIIB signal transduction pathway. In yetanother particular embodiment, the antagonistic antibodies of theinvention decrease phosphorylation of FcγRIIB or SHIP recruitment. In afurther embodiment of the invention, the antagonistic antibodies enhanceMAP kinase activity or Akt recruitment in the B cell receptor mediatedsignaling pathway. In another embodiment, the antagonistic antibodies ofthe invention antagonize FcγRIIB-mediated inhibition of FcεRI signaling.In a particular embodiment, the antagonistic antibodies of the inventionenhance FcεRI-induced mast cell activation, calcium mobilization,degranulation, cytokine production, or serotonin release. In anotherembodiment, the antagonistic antibodies of the invention inhibitphosphorylation of FcγRIIB, inhibit recruitment of SHIP, inhibit SHIPphosphorylation and its association with Shc, enhance activation of MAPkinase family members (e.g., Erk1, Erk2, JNK, p38, etc.). In yet anotherembodiment, the antagonistic antibodies of the invention inhibittyrosine phosphorylation of p62dok and its association with SHIP andrasGAP. In another embodiment, the antagonistic antibodies of theinvention enhance FcγR-mediated phagocytosis in monocytes ormacrophages. In another embodiment, the antagonistic antibodies of theinvention prevent phagocytosis, clearance of opsonized particles bysplenic macrophages.

Antibodies of the invention include, but are not limited to, monoclonalantibodies, synthetic antibodies, recombinantly produced antibodies,multispecific antibodies, human antibodies, humanized antibodies,chimeric antibodies, camelized antibodies, single-chain Fvs (scFv),single chain antibodies, Fab fragments, F(ab′) fragments,disulfide-linked Fvs (sdFv), intrabodies, and epitope-binding fragmentsof any of the above. In particular, antibodies used in the methods ofthe present invention include immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds to FcγRIIB with greater affinity than said immunoglobulin moleculebinds FcγRIIA or immunospecifically binds FcγRIIB and blocks the Fcbinding domain of FcγRIIB.

The antibodies used in the methods of the invention may be from anyanimal origin including birds and mammals (e.g., human, non-humanprimate, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse,or chicken). Preferably, the antibodies are human or humanizedmonoclonal antibodies. As used herein, “human” antibodies includeantibodies having the amino acid sequence of a human immunoglobulin andinclude antibodies isolated from human immunoglobulin libraries orlibraries of synthetic human immunoglobulin coding sequences or frommice that express antibodies from human genes.

The antibodies used in the methods of the present invention may bemonospecific, bispecific, trispecific or of greater multispecificity.Multispecific antibodies may immunospecifically bind to differentepitopes of FcγRIIB or immunospecifically bind to both an epitope ofFcγRIIB as well a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., InternationalPublication Nos. WO 93/17715, WO 92/08802, WO 91/00360, and WO 92/05793;Tutt, et al. (1991) “Trispecific F(ab′)3 Derivatives That UseCooperative Signaling Via The TCR/CD3 Complex And CD2 To Activate AndRedirect Resting Cytotoxic T Cells,” J. Immunol. 147:60-69; U.S. Pat.Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, and 5,601,819; andKostelny et al. (1992) “Formation Of A Bispecific Antibody By The Use OfLeucine Zippers,” J. Immunol. 148:1547-1553; Todorovska et al. (2001)“Design And Application Of Diabodies, Triabodies And Tetrabodies ForCancer Targeting,” Journal of Immunological Methods, 248:47-66.

In particular embodiments, the antibodies of the invention aremulti-specific with specificities for FcγRIIB and for a cancer antigenor any other cell surface marker specific for a cell designed to bekilled, e.g., in treating or preventing a particular disease ordisorder, or for other Fc receptors, e.g., FcγRIIIA, FcγRIIIB, etc.

In a specific embodiment, an antibody used in the methods of the presentinvention is an antibody or an antigen-binding fragment thereof (e.g.,comprising one or more complementarily determining regions (CDRs),preferably all 6 CDRs) of the antibody produced by clone 2B6 or 3H7 withATCC accession numbers PTA-4591 and PTA-4592, respectively (e.g., theheavy chain CDR3). In another embodiment, an antibody used in themethods of the present invention binds to the same epitope as the mousemonoclonal antibody produced from clone 2B6 or 3H7 with ATCC accessionnumbers PTA-4591 and PTA-4592, respectively and/or competes with themouse monoclonal antibody produced from clone 2B6 or 3H7 with ATCCaccession numbers PTA-4591 and PTA-4592, respectively as determined,e.g., in an ELISA assay or other appropriate competitive immunoassay,and also binds FcγRIIB with a greater affinity than said antibody or afragment thereof binds FcγRIIA.

The antibodies used in the methods of the invention include derivativesthat are modified, i.e, by the covalent attachment of any type ofmolecule to the antibody such that covalent attachment. For example, butnot by way of limitation, the antibody derivatives include antibodiesthat have been modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. Any of numerous chemical modifications maybe carried out by known techniques, including, but not limited to,specific chemical cleavage, acetylation, formylation, metabolicsynthesis of tunicamycin, etc. Additionally, the derivative may containone or more non-classical amino acids.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use human, chimeric orhumanized antibodies. Completely human antibodies are particularlydesirable for therapeutic treatment of human subjects. Human antibodiescan be made by a variety of methods known in the art including phagedisplay methods described above using antibody libraries derived fromhuman immunoglobulin sequences. (See also U.S. Pat. Nos. 4,444,887 and4,716,111; and International Publication Nos. WO 98/46645, WO 98/50433,WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741;each of which is incorporated herein by reference in its entirety.)

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of theJ_(H) region prevents endogenous antibody production. The modifiedembryonic stem cells are expanded and microinjected into blastocysts toproduce chimeric mice. The chimeric mice are then bred to producehomozygous offspring which express human antibodies. The transgenic miceare immunized using conventional methodologies with a selected antigen,e.g., all or a portion of a polypeptide of the invention. Monoclonalantibodies directed against the antigen can be obtained from theimmunized, transgenic mice using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA, IgM and IgEantibodies. For an overview of this technology for producing humanantibodies, see Lonberg et al. (1995) “Human Antibodies From TransgenicMice,” Int. Rev. Immunol. 13:65-93, which is incorporated herein byreference in its entirety. For a detailed discussion of this technologyfor producing human antibodies and human monoclonal antibodies andprotocols for producing such antibodies, see, e.g., InternationalPublication Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S.Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016,5,545,806, 5,814,318, and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.) and Medarex (Princeton, N.J.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology similar to that described above.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules such asantibodies having a variable region derived from a non-human antibodyand a human immunoglobulin constant region. Methods for producingchimeric antibodies are known in the art. (See e.g., Morrison (1985)“Transfectomas Provide Novel Chimeric Antibodies,” Science229:1202-1207; Oi et al. (1986) “Chimeric Antibodies,” BioTechniques4:214-221; Gillies et al. (1989) “High-Level Expression Of ChimericAntibodies Using Adapted Cdna Variable Region Cassettes,” J. Immunol.Methods 125:191-202; Gillies et al. (1989) “High-Level Expression OfChimeric Antibodies Using Adapted Cdna Variable Region Cassettes,” J.Immunol. Methods 125:191-202; and U.S. Pat. Nos. 6,311,415, 5,807,715,4,816,567, and 4,816,397, which are incorporated herein by reference intheir entirety.) Chimeric antibodies comprising one or more CDRs from anon-human species and framework regions from a human immunoglobulinmolecule can be produced using a variety of techniques known in the artincluding, for example, CDR-grafting (EP 239,400; InternationalPublication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101,and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan(1991) “A Possible Procedure For Reducing The Immunogenicity Of AntibodyVariable Domains While Preserving Their Ligand-Binding Properties,”Molecular Immunology 28(4/5):489-498; Studnicka et al. (1994)“Human-Engineered Monoclonal Antibodies Retain Full Specific BindingActivity By Preserving Non-CDR Complementarity-Modulating Residues,”Protein Engineering 7:805-814; and Roguska et al. (1994) “HumanizationOf Murine Monoclonal Antibodies Through Variable Domain Resurfacing,”Proc. Nat. Acad. Sci. 91:969-973), and chain shuffling (U.S. Pat. No.5,565,332). Each of the above-identified references is incorporatedherein by reference in its entirety.

Often, framework residues in the framework regions will be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,U.S. Pat. No. 5,585,089; and Riechmann et al. (1988) “Reshaping HumanAntibodies For Therapy,” Nature 332:323-327, which are incorporatedherein by reference in their entireties.)

A humanized antibody is an antibody, a variant or a fragment thereofwhich is capable of binding to a predetermined antigen and whichcomprises a framework region having substantially the amino acidsequence of a human immunoglobulin and a CDR having substantially theamino acid sequence of a non-human immunoglobulin. A humanized antibodycomprises substantially all of at least one, and typically two, variabledomains in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin (i.e., donor antibody) and all orsubstantially all of the framework regions are those of a humanimmunoglobulin consensus sequence. Preferably, a humanized antibody alsocomprises at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. Ordinarily, the antibody willcontain both the light chain as well as at least the variable domain ofa heavy chain. The antibody also may include the CHI, hinge, CH2, CH3,and CH4 regions of the heavy chain. The humanized antibody can beselected from any class of immunoglobulins, including IgM, IgG, IgD, IgAand IgE, and any isotype, including IgG₁, IgG₂, IgG₃ and IgG₄. Usuallythe constant domain is a complement fixing constant domain where it isdesired that the humanized antibody exhibit cytotoxic activity, and theclass is typically IgG₁. Where such cytotoxic activity is not desirable,the constant domain may be of the IgG₂ class. The humanized antibody maycomprise sequences from more than one class or isotype, and selectingparticular constant domains to optimize desired effector functions iswithin the ordinary skill in the art. The framework and CDR regions of ahumanized antibody need not correspond precisely to the parentalsequences, e.g., the donor CDR or the consensus framework may bemutagenized by substitution, insertion or deletion of at least oneresidue so that the CDR or framework residue at that site does notcorrespond to either the consensus or the import antibody. Suchmutations, however, will not be extensive. Usually, at least 75% of thehumanized antibody residues will correspond to those of the parentalframework region (FR) and CDR sequences, more often 90%, and mostpreferably greater than 95%. Humanized antibodies can be produced usingvariety of techniques known in the art, including but not limited to,CDR-grafting (European Patent No. EP 239,400; International PublicationNo. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and5,585,089), veneering or resurfacing (European Patent Nos. EP 592,106and EP 519,596; Padlan (1991) “A Possible Procedure For Reducing TheImmunogenicity Of Antibody Variable Domains While Preserving TheirLigand-Binding Properties,” Molecular Immunology 28(4/5):489-498;Studnicka et al. (1994) “Human-Engineered Monoclonal Antibodies RetainFull Specific Binding Activity By Preserving Non-CdrComplementarity-Modulating Residues,” Protein Engineering 7:805-814; andRoguska et al. (1994) “Humanization Of Murine Monoclonal AntibodiesThrough Variable Domain Resurfacing,” Proc. Nat. Acad. Sci. 91:969-973),chain shuffling (U.S. Pat. No. 5,565,332), and techniques disclosed in,e.g., U.S. Pat. Nos. 6,407,213, 5,766,886, 5,585,089, InternationalPublication No. WO 9317105, Tan et al. (2002) “‘Superhumanized’Antibodies: Reduction Of Immunogenic Potential ByComplementarity-Determining Region Grafting With Human GermlineSequences: Application To An Anti-CD28,” J. Immunol. 169:1119 1125;Caldas et al. (2000) “Design And Synthesis Of Germline-BasedHemi-Humanized Single-Chain Fv Against The CD18 Surface Antigen,”Protein Eng. 13:353 360; Morea et al. (2000) “Antibody Modeling:Implications For Engineering And Design,” Methods 20:267 279; Baca etal. (1997) “Antibody Humanization Using Monovalent Phage Display,” J.Biol. Chem. 272:10678-10684; Roguska et al. (1996) “A Comparison Of TwoMurine Monoclonal Antibodies Humanized By CDR-Grafting And VariableDomain Resurfacing,” Protein Eng. 9:895 904; Couto et al. (1995)“Designing Human Consensus Antibodies With Minimal PositionalTemplates,” Cancer Res. 55 (23 Supp):5973s 5977s; Couto et al. (1995)“Anti-BA46 Monoclonal Antibody Mc3: Humanization Using A NovelPositional Consensus And In Vivo And In Vitro Characterization,” CancerRes. 55:1717 22; Sandhu (1994) “A Rapid Procedure For The HumanizationOf Monoclonal Antibodies,” Gene 150:409 410; Pedersen et al. (1994)“Comparison Of Surface Accessible Residues In Human And MurineImmunoglobulin Fv Domains. Implication For Humanization Of MurineAntibodies,” J. Mol. Biol. 235:959 973; Jones et al. (1986) “ReplacingThe Complementarity-Determining Regions In A Human Antibody With ThoseFrom A Mouse,” Nature 321:522-525; Riechmann et al. (1988) “ReshapingHuman Antibodies For Therapy,” Nature 332:323-327; and Presta (1992)“Antibody Engineering,” Curr. Op. Biotech. 3:394-398. Often, frameworkresidues in the framework regions will be substituted with thecorresponding residue from the CDR donor antibody to alter, preferablyimprove, antigen binding. These framework substitutions are identifiedby methods well known in the art, e.g., by modeling of the interactionsof the CDR and framework residues to identify framework residuesimportant for antigen binding and sequence comparison to identifyunusual framework residues at particular positions. (See, e.g., U.S.Pat. No. 5,585,089; and Riechmann et al. (1988) “Reshaping HumanAntibodies For Therapy,” Nature 332:323-327, which are incorporatedherein by reference in their entireties.)

Preferably the humanized antibodies of the invention bind theextracellular domain of native human FcγRIIB. The humanized anti-FcγRIIBantibodies of the invention may have a heavy chain variable regioncomprising the amino acid sequence of CDR1 (following heavy chain CDR1amino acid sequence: NYWIH (SEQ ID NO:1); or DAWMD (SEQ ID NO:29);and/or the following heavy chain CDR2 amino acid sequence:VIDPSDTYPNYNKKFKG (SEQ ID NO:2); or EIRNKANNLATYYAESVKG (SEQ ID NO:30);and/or the following heavy chain CDR3 amino acid sequence: NGDSDYYSGMDY(SEQ ID NO:3); or YSPFAY (SEQ ID NO:31); and/or a light chain variableregion comprising the following light chain CDR1 amino acid sequence:RTSQSIGTNIH (SEQ ID NO:8); or RASQEISGYLS (SEQ ID NO:38); and/or thefollowing light chain CDR2 amino acid sequence: NVSESIS (SEQ ID NO:9);or YVSESIS (SEQ ID NO:10); or YASESIS (SEQ ID NO:11); or AASTLDS (SEQ IDNO:39); and/or the following light chain CDR3 amino acid sequence:QQSNTWPFT (SEQ ID NO:12); or LQYVSYPYT (SEQ ID NO:40).

In certain embodiments, the humanized antibodies of the inventioncomprise a light chain variable regions comprising the amino acidsequence of:

SEQ ID NO: 18: EIVLTQSPDF QSVTPKEKVT ITCRTSQSIG TNIHWYQQKP DQSPKLLIKN 50 VSESISGVPS RFSGSGSGTD FTLTINSLEA EDAATYYCQQ SNTWPFTFGG 100 GTKVEIK107;a light chain variable region comprising the amino acid sequence of:

SEQ ID NO: 29: EIVLTQSPDF QSVTPKEKVT ITCRTSQSIG TNIHWYQQKP DQSPKLLIKY 50 VSESISGVPS RFSGSGSGTD FTLTINSLEA EDAATYYCQQ SNTWPFTFGG 100 GTKVEIK107;a light chain variable region comprising the amino acid sequence of:

SEQ ID NO: 22: EIVLTQSPDF QSVTPKEKVT ITCRTSQSIG TNIHWYQQKP DQSPKLLIKY 50 ASESISGVPS RFSGSGSGTD FTLTINSLEA EDAATYCQQS NTWPFTFGGG 100 TKVEIK106; ora light chain variable region comprising the amino acid sequence of:

SEQ ID NO: 46: DIQMTQSPSS LSASLGERVS LTCRASQEIS GYLSWLQQKP DGTTRRLIYA 50 ASTLDSGVPK RFSGSWSGSD YSLTISSLES EDFADYYCLQ YVSYPYTFGG 100 GTKLEIK107;and/or a heavy chain variable region comprising the amino acid sequenceof:

SEQ ID NO: 24: QVQLVQSGAE VKKPGASVKV SCKASGYTFT NYWIHWVRQA PGQGLEWMGV 50 IDPSDTYPNY NKKFKGRVTM TTDTSTSTAY MELRSLRSDD TAVYYCARNG 100DSDYYSGMDY WGQGTTVTVS S 121; ora heavy chain variable region comprising the amino acid sequence of:

SEQ ID NO: 37: EVKFEESGGG LVQPGGSMKL SCAASGFTFS DAWMDWVRQG PEKGLEWVAE 50 IRNKANNLAT YYAESVKGRF TIPRDDSKSS VYLHMNSLRA EDTGIYYCYS 100PFAYWGQGTL VTVSA 115;and/or amino acid sequence variants thereof.

In specific embodiments, the invention encompasses a humanized antibodycomprising the CDRs of 2B6 or of 3H7. In particular, an antibody withthe heavy chain variable domain having the amino acid sequence of SEQ IDNO:24 and the light chain variable domain having the amino acid sequenceof SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22. In a specificembodiment, the invention encompasses a humanized antibody with theheavy chain variable domain having the amino acid sequence of SEQ IDNO:37 and the light chain variable domain having the amino acid sequenceof SEQ ID NO:46.

In one specific embodiment, the invention provides a humanized 2B6antibody, wherein the VH region consists of the FR segments from thehuman germline VH segment VH1-18 (Matsuda et al. (1998) “The CompleteNucleotide Sequence Of The Human Immunoglobulin Heavy Chain VariableRegion Locus,” J. Exp. Med. 188:2151-2162) and JH6 (Ravetch et al.(1981) “Structure Of The Human Immunoglobulin Mu Locus: CharacterizationOf Embryonic And Rearranged J And D Genes,” Cell 27(3 Pt. 2): 583-591),and one or more CDR regions of the 2B6 VH, having the amino acidsequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In one embodiment,the 2B6 VH has the amino acid sequence of SEQ ID NO:24. In anotherspecific embodiment, the humanized 2B6 antibody further comprises a VLregion, which consists of the FR segments of the human germline VLsegment VK-A26 (Lautner-Rieske et al. (1992) “The Human ImmunoglobulinKappa Locus. Characterization Of The Duplicated A Regions,” Eur. J.Immunol. 22:1023-1029) and JK4 (Hieter et al. (1982) “Evolution Of HumanImmunoglobulin Kappa J Region Genes,” J. Biol. Chem. 257:1516-1522), andone or more CDR regions of 2B6 VL, having the amino acid sequence of SEQID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12. Inone embodiment, the 2B6 VL has the amino acid sequence of SEQ ID NO:18;SEQ ID NO:20, or SEQ ID NO:22.

In another specific embodiment, the invention provides a humanized 3H7antibody, wherein the VH region consists of the FR segments from a humangermline VH segment and the CDR regions of the 3H7 VH, having the aminoacid sequence of SEQ ID NO:37. In another specific embodiment, thehumanized 3H7 antibody further comprises a VL regions, which consists ofthe FR segments of a human germline VL segment and the CDR regions of3H7VL, having the amino acid sequence of SEQ ID NO:46.

In particular, the invention provides a humanized antibody thatimmunospecifically binds to extracellular domain of native humanFcγRIIB, said antibody comprising (or alternatively, consisting of) CDRsequences of 2B6 or 3H7, in any of the following combinations: a VH CDR1and a VL CDR1; a VH CDR1 and a VL CDR2; a VH CDR1 and a VL CDR3; a VHCDR2 and a VL CDR1; VH CDR2 and VL CDR2; a VH CDR2 and a VL CDR3; a VHCDR3 and a VH CDR1; a VH CDR3 and a VL CDR2; a VH CDR3 and a VL CDR3; aVH1 CDR1, a VH CDR2 and a VL CDR1; a VH CDR1, a VH CDR2 and a VL CDR2; aVH CDR1, a VH CDR2 and a VL CDR3; a VH CDR2, a VH CDR3 and a VL CDR1, aVH CDR2, a VH CDR3 and a VL CDR2; a VH CDR2, a VH CDR2 and a VL CDR3; aVH CDR1, a VL CDR1 and a VL CDR2; a VH CDR1, a VL CDR1 and a VL CDR3; aVH CDR2, a VL CDR1 and a VL CDR2; a VH CDR2, a VL CDR1 and a VL CDR3; aVH CDR3, a VL CDR1 and a VL CDR2; a VH CDR3, a VL CDR1 and a VL CDR3; aVH CDR1, a VH CDR2, a VH CDR3 and a VL CDR1; a VH CDR1, a VH CDR2, a VHCDR3 and a VL CDR2; a VH CDR1, a VH CDR2, a VH CDR3 and a VL CDR3; a VHCDR1, a VH CDR2, a VL CDR1 and a VL CDR2; a VH CDR1, a VH CDR2, a VLCDR1 and a VL CDR3; a VH CDR1, a VH CDR3, a VL CDR1 and a VL CDR2; a VHCDR1, a VH CDR3, a VL CDR1 and a VL CDR3; a VH CDR2, a VH CDR3, a VLCDR1 and a VL CDR2; a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR3; a VHCDR2, a VH CDR3, a VL CDR2 and a VL CDR3; a VH CDR1, a VH CDR2, a VHCDR3, a VL CDR1 and a VL CDR2; a VH CDR1, a VH CDR2, a VH CDR3, a VLCDR1 and a VL CDR3; a VH CDR1, a VH CDR2, a VL CDR1, a VL CDR2, and a VLCDR3; a VH CDR1, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3; a VHCDR2, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3; or any combinationthereof of the VH CDRs and VL CDRs disclosed herein.

In some embodiments, at least one CDR from the donor antibody is graftedonto the human antibody. In other embodiments, at least two andpreferably all three CDRs of each of the heavy and/or light chainvariable regions are grafted onto the human antibody. The CDRs maycomprise the Kabat CDRs, the structural loop CDRs or a combinationthereof. In some embodiments, the invention encompasses a humanizedFcγRIIB antibody comprising at least one CDR grafted heavy chain and atleast one CDR-grafted light chain.

Further, the antibodies of the invention can, in turn, be utilized togenerate anti-idiotype antibodies using techniques well known to thoseskilled in the art. (See, e.g., Greenspan et al. (1989) “CooperativeBinding Of Two Antibodies To Independent Antigens By An Fc-DependentMechanism,” FASEB J. 7:437-444; and Nissinoff (1991) “Idiotypes:Concepts And Applications,” J. Immunol. 147:2429-2438). The inventionprovides methods employing the use of polynucleotides comprising anucleotide sequence encoding an antibody of the invention or a fragmentthereof.

The present invention encompasses single domain antibodies, includingcamelized single domain antibodies (See e.g., Muyldermans et al. (2001)“Recognition Of Antigens By Single-Domain Antibody Fragments: TheSuperfluous Luxury Of Paired Domains,” Trends Biochem. Sci. 26:230-235;Nuttall et al. (2000) “Immunoglobulin VH Domains And Beyond: Design AndSelection Of Single-Domain Binding And Targeting Reagents,” Cur. Pharm.Biotech. 1:253-263; Reichmann et al. (1999) “Single Domain Antibodies:Comparison Of Camel VH And Camelised Human VH Domains,” J. Immunol.Meth. 231:25-38; International Publication Nos. WO 94/04678 and WO94/25591; U.S. Pat. No. 6,005,079; which are incorporated herein byreference in their entireties). In one embodiment, the present inventionprovides single domain antibodies comprising two VH domains withmodifications such that single domain antibodies are formed.

The methods of the present invention also encompass the use ofantibodies or fragments thereof that have half-lives (e.g., serumhalf-lives) in a mammal, preferably a human, of greater than 15 days,preferably greater than 20 days, greater than 25 days, greater than 30days, greater than 35 days, greater than 40 days, greater than 45 days,greater than 2 months, greater than 3 months, greater than 4 months, orgreater than 5 months. The increased half-lives of the antibodies of thepresent invention or fragments thereof in a mammal, preferably a human,results in a higher serum titer of said antibodies or antibody fragmentsin the mammal, and thus, reduces the frequency of the administration ofsaid antibodies or antibody fragments and/or reduces the concentrationof said antibodies or antibody fragments to be administered. Antibodiesor fragments thereof having increased in vivo half-lives can begenerated by techniques known to those of skill in the art. For example,antibodies or fragments thereof with increased in vivo half-lives can begenerated by modifying (e.g., substituting, deleting or adding) aminoacid residues identified as involved in the interaction between the Fcdomain and the FcRn receptor. The antibodies of the invention may beengineered by methods described in Ward et al. to increase biologicalhalf-lives (See U.S. Pat. No. 6,277,375 B1). For example, antibodies ofthe invention may be engineered in the Fc-hinge domain to have increasedin vivo or serum half-lives.

Antibodies or fragments thereof with increased in vivo half-lives can begenerated by attaching to said antibodies or antibody fragments polymermolecules such as high molecular weight polyethyleneglycol (PEG). PEGcan be attached to said antibodies or antibody fragments with or withouta multifunctional linker, either through site-specific conjugation ofthe PEG to the N- or C-terminus of said antibodies or antibody fragmentsor via epsilon-amino groups present on lysine residues. Linear orbranched polymer derivatization that results in minimal loss ofbiological activity will be used. The degree of conjugation will beclosely monitored by SDS-PAGE and mass spectrometry to ensure properconjugation of PEG molecules to the antibodies. Unreacted PEG can beseparated from antibody-PEG conjugates by, e.g., size exclusion orion-exchange chromatography.

The antibodies of the invention may also be modified by the methods andcoupling agents described by Davis et al. (See U.S. Pat. No. 4,179,337)in order to provide compositions that can be injected into the mammaliancirculatory system with substantially no immunogenic response.

The present invention also encompasses the use of antibodies or antibodyfragments comprising the amino acid sequence of any of the antibodies ofthe invention with mutations (e.g., one or more amino acidsubstitutions) in the framework or variable regions. Preferably,mutations in these antibodies maintain or enhance the avidity and/oraffinity of the antibodies for the particular antigen(s) to which theyimmunospecifically bind. Standard techniques known to those skilled inthe art (e.g., immunoassays) can be used to assay the affinity of anantibody for a particular antigen.

The present invention encompasses antibodies comprising modificationspreferably, in the Fc region that modify the binding affinity of theantibody to one or more FcγR. Methods for modifying antibodies withmodified binding to one or more FcγR are known in the art, see, e.g.,PCT Publication Nos. WO 99/58572, WO 99/51642, WO 98/23289, WO 89/07142,WO 88/07089, and U.S. Pat. Nos. 5,843,597 and 5,642,821, each of whichis incorporated herein by reference in their entirety. The inventionencompasses any of the mutations disclosed in U.S. Application Nos.60/439,498 and 60/456,041, filed Jan. 9, 2003 and Mar. 19, 2003,respectively each of which is incorporated herein by reference in theirentirety. In some embodiments, the invention encompasses antibodies thathave altered affinity for an activating FcγR, e.g., FcγRIIIA.Preferably, such modifications also have an altered Fc-mediated effectorfunction. Modifications that affect Fc-mediated effector function arewell known in the art (See U.S. Pat. No. 6,194,551, which isincorporated herein by reference in its entirety). The amino acids thatcan be modified in accordance with the method of the invention includebut are not limited to Proline 329, Proline 331, and Lysine 322. Proline329, 331 and Lysine 322 are preferably replaced with alanine, however,substitution with any other amino acid is contemplated. SeeInternational Publication No. WO 00/42072 and U.S. Pat. No. 6,194,551,which are incorporated herein by reference in their entirety.

In one particular embodiment, the modification of the Fc regioncomprises one or more mutations in the Fc region. The one or moremutations in the Fc region may result in an antibody with an alteredantibody-mediated effector function, an altered binding to other Fcreceptors (e.g., Fc activation receptors), an altered ADCC activity, oran altered C1q binding activity, or an altered complement dependentcytotoxicity activity, or any combination thereof.

The invention also provides antibodies with altered oligosaccharidecontent. Oligosaccharides as used herein refer to carbohydratescontaining two or more simple sugars and the two terms may be usedinterchangeably herein. Carbohydrate moieties of the instant inventionwill be described with reference to commonly used nomenclature in theart. For a review of carbohydrate chemistry, see, e.g., Hubbard et al.(1981) “Synthesis And Processing Of Asparagine-Linked Oligosaccharides,”Ann. Rev. Biochem., 50: 555-583, which is incorporated herein byreference in its entirety. This nomenclature includes for example, Man,which represents mannose; GlcNAc which represents 2-N-acetylglucosamine;Gal which represents galactose; Fuc for fucose and Glc for glucose.Sialic acids are described by the shorthand notation NeuNAc for5-N-acetylneuraminic acid, and NeuNGc for 5-glycolneuraminic.

In general, antibodies contain carbohydrate moeities at conservedpositions in the constant region of the heavy chain, and up to 30% ofhuman IgGs have a glycosylated Fab region. IgG has a single N-linkedbiantennary carbohydrate structure at Asn 297 which resides in the CH2domain (Jefferis et al. (1998) “IgG-Fc-Mediated Effector Functions:Molecular Definition Of Interaction Sites For Effector Ligands And TheRole Of Glycosylation,” Immunol. Rev. 163: 59-76; Wright et al. (1997)“Effect Of Glycosylation On Antibody Function: Implications For GeneticEngineering,” Trends Biotech. 15: 26-32). Human IgG typically has acarbohydrate of the following structure;GlcNAc(Fucose)-GlcNAc-Man-(ManGlcNAc)₂. However variations among IgGs incarbohydrate content does occur which leads to altered function, see,e.g., Jassal et al. (2001) “Sialylation Of Human IgG-Fc Carbohydrate ByTransfected Rat Alpha2,6-Sialyltransferas,” Biochem. Biophys. Res.Commun. 288: 243-249; Groenink et al. (1996) “On The Interaction BetweenAgalactosyl IgG And Fc Gamma Receptors,” Eur. J. Immunol. 26: 1404-1407;Boyd et al. (1995) “The Effect Of The Removal Of Sialic Acid, GalactoseAnd Total Carbohydrate On The Functional Activity Of Campath-1H,” Mol.Immunol. 32: 1311-1318; Kumpel et al. (1994) “Galactosylation Of HumanIgG Monoclonal Anti-D Produced By EBV-Transformed B-Lymphoblastoid CellLines Is Dependent On Culture Method And Affects Fc Receptor-MediatedFunctional Activity,” Human Antibody Hybridomas, 5: 143-151. Theinvention encompasses antibodies comprising a variation in thecarbohydrate moiety that is attached to Asn 297. In one embodiment, thecarbohydrate moiety has a galactose and/or galactose-sialic acid at oneor both of the terminal GlcNAc and/or a third GlcNac arm (bisectingGlcNAc).

In some embodiments, the antibodies of the invention are substantiallyfree of one or more selected sugar groups, e.g., one or more sialic acidresidues, one or more galactose residues, one or more fucose residues.An antibody that is substantially free of one or more selected sugargroups may be prepared using common methods known to one skilled in theart, including for example recombinantly producing an antibody of theinvention in a host cell that is defective in the addition of theselected sugar groups(s) to the carbohydrate moiety of the antibody,such that about 90-100% of the antibody in the composition lacks theselected sugar group(s) attached to the carbohydrate moiety. Alternativemethods for preparing such antibodies include for example, culturingcells under conditions that prevent or reduce the addition of one ormore selected sugar groups, or post-translational removal of one or moreselected sugar groups.

In a specific embodiment, the invention encompasses a method ofproducing a substantially homogenous antibody preparation, wherein about80-100% of the antibody in the composition lacks a fucose on itscarbohydrate moiety, e.g., the carbohydrate attachment on Asn 297. Theantibody may be prepared, for example, by: (A) use of an engineered hostcell that is deficient in fucose metabolism such that it has a reducedability to fucosylate proteins expressed therein; (B) culturing cellsunder conditions which prevent or reduce fusocylation; (C)post-translational removal of fucose, e.g., with a fucosidase enzyme; or(D) purification of the antibody so as to select for the product whichis not fucosylated. Most preferably, nucleic acid encoding the desiredantibody is expressed in a host cell that has a reduced ability tofucosylate the antibody expressed therein. Preferably the host cell is adihydrofolate reductase deficient chinese hamster ovary cell (CHO),e.g., a Lec 13 CHO cell (lectin resistant CHO mutant cell line; Ripka etal. (1986) “Lectin-Resistant CHO Cells: Selection Of Four New PeaLectin-Resistant Phenotypes,” Somatic Cell & Molec. Gen. 12(1): 51-62;Ripka et al. (1986) “Two Chinese Hamster Ovary Glycosylation MutantsAffected In The Conversion Of GDP-Mannose To GDP-Fucose,” Arch. Biochem.Biophys. 249(2): 533-545), CHO-K1, DUX-B11, CHO-DP12 or CHO-DG44, whichhas been modified so that the antibody is not substantially fucosylated.Thus, the cell may display altered expression and/or activity for thefucoysltransferase enzyme, or another enzyme or substrate involved inadding fucose to the N-linked oligosaccharide so that the enzyme has adiminished activity and/or reduced expression level in the cell. Formethods to produce antibodies with altered fucose content, see, e.g., WO03/035835 and Shields et al. (2002) “Lack Of Fucose On Human IgG1N-Linked Oligosaccharide Improves Binding To Human Fcgamma RIII AndAntibody-Dependent Cellular Toxicity,” J. Biol. Chem. 277(30):26733-26740; both of which are incorporated herein by reference in theirentirety.

In some embodiments, the altered carbohydrate modifications modulate oneor more of the following: solubilization of the antibody, facilitationof subcellular transport and secretion of the antibody, promotion ofantibody assembly, conformational integrity, and antibody-mediatedeffector function. In a specific embodiment, the altered carbohydratemodifications enhance antibody mediated effector function relative tothe antibody lacking the carbohydrate modification. Carbohydratemodifications that lead to altered antibody mediated effector functionare well known in the art (see e.g., Shields et al. (2002) “Lack OfFucose On Human IgG1 N-Linked Oligosaccharide Improves Binding To HumanFcgamma RIII And Antibody-Dependent Cellular Toxicity,” J. Biol. Chem.277(30): 26733-26740; Davies et al. (2001) “Expression Of GnTIII In ARecombinant Anti-CD20 CHO Production Cell Line: Expression Of AntibodiesWith Altered Glycoforms Leads To An Increase In ADCC Through HigherAffinity For FCγRIII,” Biotechnology & Bioengineering, 74(4): 288-294).In another specific embodiment, the altered carbohydrate modificationsenhance the binding of antibodies of the invention to FcγRIIB receptor.Altering carbohydrate modifications in accordance with the methods ofthe invention includes, for example, increasing the carbohydrate contentof the antibody or decreasing the carbohydrate content of the antibody.Methods of altering carbohydrate contents are known to those skilled inthe art, see, e.g., Wallick et al. (1988) “Glycosylation Of A VH ResidueOf A Monoclonal Antibody Against Alpha (1 - - - 6) Dextran Increases ItsAffinity For Antigen,” Journal of Exp. Med. 168(3): 1099-1109; Tao etal. (1989) “Studies Of Aglycosylated Chimeric Mouse-Human IgG. Role OfCarbohydrate In The Structure And Effector Functions Mediated By TheHuman IgG Constant Region,” J. Immunol., 143(8): 2595-2601; Routledge etal. (1995) “The Effect Of Aglycosylation On The Immunogenicity Of AHumanized Therapeutic CD3 Monoclonal Antibody,” Transplantation, 60(8):847-853; Elliott et al. (2003) “Enhancement Of Therapeutic Protein InVivo Activities Through Glycoengineering,” Nature Biotechnology, 21:414-421; Shields et al. (2002) “Lack Of Fucose On Human IgG1 N-LinkedOligosaccharide Improves Binding To Human Fcgamma RIII AndAntibody-Dependent Cellular Toxicity,” J. Biol. Chem. 277(30):26733-26740; all of which are incorporated herein by reference in theirentirety.

In some embodiments, the invention encompasses antibodies comprising oneor more glycosylation sites, so that one or more carbohydrate moietiesare covalently attached to the antibody. In other embodiments, theinvention encompasses antibodies comprising one or more glycosylationsites and one or more modifications in the Fc region, such as thosedisclosed supra and those known to one skilled in the art. In preferredembodiments, the one or more modifications in the Fc region enhance theaffinity of the antibody for an activating FcγR, e.g., FcγRIIIA,relative to the antibody comprising the wild type Fc regions. Antibodiesof the invention with one or more glycosylation sites and/or one or moremodifications in the Fc region have an enhanced antibody mediatedeffector function, e.g., enhanced ADCC activity. In some embodiments,the invention further comprises antibodies comprising one or moremodifications of amino acids that are directly or indirectly known tointeract with a carbohydrate moiety of the antibody, including but notlimited to amino acids at positions 241, 243, 244, 245, 245, 249, 256,258, 260, 262, 264, 265, 296, 299, and 301. Amino acids that directly orindirectly interact with a carbohydrate moiety of an antibody are knownin the art, see, e.g., Jefferis et al. (1995) “Recognition Sites OnHuman IgG For Fc Gamma Receptors The Role Of Glycosylation,” ImmunologyLetters, 44: 111-117, which is incorporated herein by reference in itsentirety.

The invention encompasses antibodies that have been modified byintroducing one or more glycosylation sites into one or more sites ofthe antibodies, preferably without altering the functionality of theantibody, e.g., binding activity to FcγRIIB. Glycosylation sites may beintroduced into the variable and/or constant region of the antibodies ofthe invention. As used herein, “glycosylation sites” include anyspecific amino acid sequence in an antibody to which an oligosaccharide(i.e., carbohydrates containing two or more simple sugars linkedtogether) will specifically and covalently attach. Oligosaccharide sidechains are typically linked to the backbone of an antibody via either N-or O-linkages. N-linked glycosylation refers to the attachment of anoligosaccharide moiety to the side chain of an asparagine residue.O-linked glycosylation refers to the attachment of an oligosaccharidemoiety to a hydroxyamino acid, e.g., serine, threonine. The antibodiesof the invention may comprise one or more glycosylation sites, includingN-linked and O-linked glycosylation sites. Any glycosylation site forN-linked or O-linked glycosylation known in the art may be used inaccordance with the instant invention. An exemplary N-linkedglycosylation site that is useful in accordance with the methods of thepresent invention, is the amino acid sequence: Asn-X-Thr/Ser, wherein Xmay be any amino acid and Thr/Ser indicates a threonine or a serine.Such a site or sites may be introduced into an antibody of the inventionusing methods well known in the art to which this invention pertains.See, for example, “In Vitro Mutagenesis,” Recombinant DNA: A ShortCourse, J. D. Watson, et al. W.H. Freeman and Company, New York, 1983,chapter 8, pp. 106-116, which is incorporated herein by reference in itsentirety. An exemplary method for introducing a glycosylation site intoan antibody of the invention may comprise modifying or mutating an aminoacid sequence of the antibody so that the desired Asn-X-Thr/Ser sequenceis obtained.

In some embodiments, the invention encompasses methods of modifying thecarbohydrate content of an antibody of the invention by adding ordeleting a glycosylation site. Methods for modifying the carbohydratecontent of antibodies are well known in the art and encompassed withinthe invention, see, e.g., U.S. Pat. No. 6,218,149; EP 0 359 096 B1; U.S.Publication No. US 2002/0028486; WO 03/035835; U.S. Publication No.2003/0115614; U.S. Pat. No. 6,218,149; U.S. Pat. No. 6,472,511; all ofwhich are incorporated herein by reference in their entirety. In otherembodiments, the invention encompasses methods of modifying thecarbohydrate content of an antibody of the invention by deleting one ormore endogenous carbohydrate moieties of the antibody.

The invention further encompasses methods of modifying an effectorfunction of an antibody of the invention, wherein the method comprisesmodifying the carbohydrate content of the antibody using the methodsdisclosed herein or known in the art.

Standard techniques known to those skilled in the art can be used tointroduce mutations in the nucleotide sequence encoding an antibody, orfragment thereof, including, e.g., site-directed mutagenesis andPCR-mediated mutagenesis, which results in amino acid substitutions.Preferably, the derivatives include less than 15 amino acidsubstitutions, less than 10 amino acid substitutions, less than 5 aminoacid substitutions, less than 4 amino acid substitutions, less than 3amino acid substitutions, or less than 2 amino acid substitutionsrelative to the original antibody or fragment thereof. In a preferredembodiment, the derivatives have conservative amino acid substitutionsmade at one or more predicted non-essential amino acid residues.

The present invention also encompasses antibodies or fragments thereofcomprising an amino acid sequence of a variable heavy chain and/orvariable light chain that is at least 45%, at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99% identical to theamino acid sequence of the variable heavy chain and/or light chain ofthe mouse monoclonal antibody produced by clone 2B6 or 3H7 having ATCCaccession numbers PTA-4591 and PTA-4592, respectively. The presentinvention further encompasses antibodies or fragments thereof thatspecifically bind FcγRIIB with greater affinity than said antibody orfragment thereof binds FcγRIIA and antibodies or a fragments thereofthat specifically binds FcγRIIB and block the Fc binding domain ofFcγRIIB, said antibodies or antibody fragments comprising an amino acidsequence of one or more CDRs that is at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to the amino acid sequence of one or more CDRs of the mousemonoclonal antibody produced by clone 2B6 or 3H7 having ATCC accessionnumbers PTA-4591 and PTA-4592, respectively. The determination ofpercent identity of two amino acid sequences can be determined by anymethod known to one skilled in the art, including BLAST proteinsearches.

The present invention also encompasses the use of antibodies or antibodyfragments that specifically bind FcγRIIB with greater affinity than saidantibodies or fragments thereof binds FcγRIIA and antibodies or antibodyfragments thereof that specifically binds FcγRIIB and block the Fcbinding domain of FcγRIIB, wherein said antibodies or antibody fragmentsare encoded by a nucleotide sequence that hybridizes to the nucleotidesequence of the mouse monoclonal antibody produced by clone 2B6 or 3H7having ATCC accession numbers PTA-4591 and PTA-4592, respectively, understringent conditions. In a preferred embodiment, the invention providesantibodies or fragments thereof that specifically bind FcγRIIB withgreater affinity than said antibodies or fragments thereof bind FcγRIIAand antibodies or a fragments thereof that specifically binds FcγRIIBand block the Fc binding domain of FcγRIIB, said antibodies or antibodyfragments comprising a variable light and/or variable heavy chainencoded by a nucleotide sequence that hybridizes under stringentconditions to the nucleotide sequence of the variable light and/orvariable heavy chain of the mouse monoclonal antibody produced by clone2B6 or 3H7 having ATCC accession numbers PTA-4591 and PTA-4592,respectively, under stringent conditions. In another preferredembodiment, the invention provides antibodies or fragments thereof thatspecifically bind FcγRIIB with greater affinity than said antibodies orfragments thereof bind FcγRIIA and antibodies or a fragments thereofthat specifically binds FcγRIIB and block the Fc binding domain ofFcγRIIB, said antibodies or antibody fragments comprising one or moreCDRs encoded by a nucleotide sequence that hybridizes under stringentconditions to the nucleotide sequence of one or more CDRs of the mousemonoclonal antibody produced by clone 2B6 or 3H7 with ATCC accessionnumbers PTA-4591 and PTA-4592, respectively. Stringent hybridizationconditions include, but are not limited to, hybridization tofilter-bound DNA in 6× sodium chloride/sodium citrate (SSC) at about 45°C. followed by one or more washes in 0.2×SSC/0.1% SDS at about 50-65°C., highly stringent conditions such as hybridization to filter-boundDNA in 6×SSC at about 45° C. followed by one or more washes in0.1×SSC/0.2% SDS at about 60° C., or any other stringent hybridizationconditions known to those skilled in the art (see, for example, Ausubel,F. M. et al., eds. 1989 Current Protocols in Molecular Biology, vol. 1,Green Publishing Associates, Inc. and John Wiley and Sons, Inc., NY atpages 6.3.1 to 6.3.6 and 2.10.3), incorporated herein by reference.

II. Antibody Conjugates

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to heterologous polypeptides (i.e., an unrelatedpolypeptide; or portion thereof, preferably at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90 or at least 100 amino acids of the polypeptide) togenerate fusion proteins. The fusion does not necessarily need to bedirect, but may occur through linker sequences. Antibodies may be usedfor example to target heterologous polypeptides to particular celltypes, either in vitro or in vivo, by fusing or conjugating theantibodies to antibodies specific for particular cell surface receptors.Antibodies fused or conjugated to heterologous polypeptides may also beused in in vitro immunoassays and purification methods using methodsknown in the art. See e.g., PCT publication Number WO 93/2 1232; EP439,095; Naramura et al. (1994) “Mechanisms Of Cellular CytotoxicityMediated By A Recombinant Antibody-IL2 Fusion Protein Against HumanMelanoma Cells,” Immunol. Lett., 39:91-99; U.S. Pat. No. 5,474,981;Gillies et al. (1992) “Antibody-Targeted Interleukin 2 Stimulates T-CellKilling Of Autologous Tumor Cells,” Proc. Nat. Acad. Sci., 89:1428-1432;and Fell et al. (1991) “Genetic Construction And Characterization Of AFusion Protein Consisting Of A Chimeric F(ab) With Specificity ForCarcinomas And Human IL-2,” J. Immunol., 146:2446-2452, which areincorporated herein by reference in their entireties.

Further, an antibody may be conjugated to a therapeutic agent or drugmoiety that modifies a given biological response. Therapeutic agents ordrug moieties are not to be construed as limited to classical chemicaltherapeutic agents. For example, the drug moiety may be a protein orpolypeptide possessing a desired biological activity. Such proteins mayinclude, for example, a toxin such as abrin, ricin A, pseudomonasexotoxin (i.e., PE-40), or diphtheria toxin, ricin, gelonin, andpokeweed antiviral protein, a protein such as tumor necrosis factor,interferons including, but not limited to, α-interferon (IFN-α),β-interferon (IFN-β), nerve growth factor (NGF), platelet derived growthfactor (PDGF), tissue plasminogen activator (TPA), an apoptotic agent(e.g., TNF-α, TNF-β, AIM I as disclosed in PCT Publication No. WO97/33899), AIM II (see, PCT Publication No. WO 97/34911), Fas Ligand(Takahashi et al. (1994) “Human Fas Ligand: Gene Structure, ChromosomalLocation And Species Specificity,” Int. Immunol., 6:1567-1574), and VEGI(PCT Publication No. WO 99/23105), a thrombotic agent or ananti-angiogenic agent (e.g., angiostatin or endostatin), or a biologicalresponse modifier such as, for example, a lymphokine (e.g.,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), andgranulocyte colony stimulating factor (“G-CSF”)), macrophage colonystimulating factor, (“M-CSF”), or a growth factor (e.g., growth hormone(“GH”); proteases, or ribonucleases.

Antibodies can be fused to marker sequences, such as a peptide tofacilitate purification. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al. (1989) “Bioassay For Trans-Activation Using Purified HumanImmunodeficiency Virus Tat-Encoded Protein: Trans-Activation RequiresmRNA Synthesis,” Proc. Natl. Acad. Sci. USA, 86:821-824, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the hemagglutinin “HA” tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al. (1984)“The Structure Of An Antigenic Determinant In A Protein,” Cell,37:767-778) and the “flag” tag (Knappik et al. (1994) “An ImprovedAffinity Tag Based On The FLAG Peptide For The Detection AndPurification Of Recombinant Antibody Fragments,” Biotechniques,17(4):754-761).

The present invention further includes compositions comprisingheterologous polypeptides fused or conjugated to antibody fragments. Forexample, the heterologous polypeptides may be fused or conjugated to aFab fragment, Fd fragment, Fv fragment, F(ab)₂ fragment, or portionthereof. Methods for fusing or conjugating polypeptides to antibodyportions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603,5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; EP 307,434;EP 367,166; International Publication Nos. WO 96/04388 and WO 91/06570;Ashkenazi et al. (1991) “Protection Against Endotoxic Shock By A TumorNecrosis Factor Receptor Immunoadhesin,” Proc. Nat. Acad. Sci. 88:10535-10539; Zheng et al. (1995) “Administration Of NoncytolyticIL-10/Fc In Murine Models Of Lipopolysaccharide-Induced Septic Shock AndAllogeneic Islet Transplantation,” J. Immunol. 154:5590-5600; and Vie etal. (1992) “Human Fusion Proteins Between Interleukin 2 And IgM HeavyChain Are Cytotoxic For Cells Expressing The Interleukin 2 Receptor,”Proc. Nat. Acad. Sci. 89:11337-11341, said references incorporated byreference in their entireties).

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of antibodies of the invention orfragments thereof (e.g., antibodies or fragments thereof with higheraffinities and lower dissociation rates). See, generally, U.S. Pat. Nos.5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten etal. (1997) “Applications Of DNA Shuffling To Pharmaceuticals AndVaccines,” Curr. Opinion Biotechnol. 8:724-733; Harayama (1998)“Artificial Evolution By DNA Shuffling,” Trends Biotechnol. 16:76-82;Hansson, et al. (1999) “Evolution Of Differential SubstrateSpecificities In Mu Class Glutathione Transferases Probed By DNAShuffling,” J. Mol. Biol. 287:265-276; and Lorenzo et al. (1998)“PCR-Based Method For The Introduction Of Mutations In Genes Cloned AndExpressed In Vaccinia Virus,” BioTechniques 24:308-313; each of thesepatents and publications are hereby incorporated by reference in itsentirety. Antibodies or fragments thereof, or the encoded antibodies orfragments thereof, may be altered by being subjected to randommutagenesis by error-prone PCR, random nucleotide insertion or othermethods prior to recombination. One or more portions of a polynucleotideencoding an antibody or antibody fragment, which portions specificallybind to FcγRIIB may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules.

The present invention also encompasses antibodies conjugated to adiagnostic or therapeutic agent or any other molecule for which serumhalf-life is desired to be increased. The antibodies can be useddiagnostically to, for example, monitor the development or progressionof a disease, disorder or infection as part of a clinical testingprocedure to, e.g., determine the efficacy of a given treatment regimen.Detection can be facilitated by coupling the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, radioactive materials, positron emittingmetals, and nonradioactive paramagnetic metal ions. The detectablesubstance may be coupled or conjugated either directly to the antibodyor indirectly, through an intermediate (such as, for example, a linkerknown in the art) using techniques known in the art. See, for example,U.S. Pat. No. 4,741,900 for metal ions that can be conjugated toantibodies for use as diagnostics according to the present invention.Such diagnosis and detection can be accomplished by coupling theantibody to detectable substances including, but not limited to, variousenzymes, enzymes including, but not limited to, horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;prosthetic group complexes such as, but not limited to,streptavidin/biotin and avidin/biotin; fluorescent materials such as,but not limited to, umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; luminescent material such as, but not limitedto, luminol; bioluminescent materials such as, but not limited to,luciferase, luciferin, and aequorin; radioactive material such as, butnot limited to, bismuth (²¹³Bi), carbon (¹⁴C), chromium (⁵¹Cr), cobalt(⁵⁷Co), fluorine (¹⁸F), gadolinium (¹⁵³Gd, ¹⁵⁹Gd), gallium (⁶⁸Ga, ⁶⁷Ga),germanium (⁶⁸Ge), holmium (¹⁶⁶Ho), indium (¹¹⁵In, ¹¹³In, ¹¹²In), iodine(¹³¹I, ¹²⁵, ¹²³I, ¹²¹I), lanthanium (¹⁴⁰La), lutetium (¹⁷⁷Lu), manganese(⁵⁴Mn), molybdenum (⁹⁹Mo), palladium (¹⁰³Pd), phosphorous (³²P),praseodymium (¹⁴²Pr), promethium (¹⁴⁹ Pm), rhenium (¹⁸⁶Re, ¹⁸⁸Re),rhodium (¹⁰⁵Rh), ruthemium (⁹⁷Ru), samarium (¹⁵³Sm), scandium (⁴⁷Sc),selenium (⁷⁵Se), strontium (⁸⁵ Sr), sulfur (³⁵S), technetium (⁹⁹Tc),thallium (²⁰¹Ti), tin (¹¹³Sn, ¹¹⁷Sn), tritium (³H), xenon (¹³³ Xe),ytterbium (¹⁶⁹Yb, ¹⁷⁵Yb), yttrium (⁹⁰Y), zinc (⁶⁵Zn); positron emittingmetals using various positron emission tomographies, and nonradioactiveparamagnetic metal ions.

An antibody may be conjugated to a therapeutic moiety such as acytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agentor a radioactive element (e.g., alpha-emitters, gamma-emitters, etc.).Cytotoxins or cytotoxic agents include any agent that is detrimental tocells. Examples include paclitaxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, BiCNU® (carmustine; BSNU) and lomustine(CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin,mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Moreover, an antibody can be conjugated to therapeutic moieties such asa radioactive materials or macrocyclic chelators useful for conjugatingradiometal ions (see above for examples of radioactive materials). Incertain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.(1998) “Comparison Of1,4,7,10-Tetraazacyclododecane-N,N′,N″,N′″-Tetraacetic Acid(DOTA)-Peptide-ChL6, A Novel Immunoconjugate With Catabolizable Linker,To 2-Iminothiolane-2-[p-(bromoacetamido)benzyl]-DOTA-ChL6 In BreastCancer Xenografts,” Clin. Cancer Res. 4:2483-2490; Peterson et al.(1999) “Enzymatic Cleavage Of Peptide-Linked Radiolabels FromImmunoconjugates,” Bioconjug. Chem. 10:553-557; and Zimmerman et al.(1999) “A Triglycine Linker Improves Tumor Uptake And BiodistributionsOf 67-Cu-Labeled Anti-Neuroblastoma MAb chCE7 F(ab′)2 Fragments,” Nucl.Med. Biol. 26:943-950 each incorporated by reference in theirentireties.

Techniques for conjugating such therapeutic moieties to antibodies arewell known; see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in MONOCLONAL ANTIBODIESAND CANCER THERAPY, Reisfeld et al. (eds.), 1985, pp. 243-256, Alan R.Liss, Inc.); Hellstrom et al., “Antibodies For Drug Delivery”, inCONTROLLED DRUG DELIVERY (2nd Ed.), Robinson et al. (eds.), 1987, pp.623-653, Marcel Dekker, Inc.); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in MONOCLONAL ANTIBODIES '84:BIOLOGICAL AND CLINICAL APPLICATIONS. Pinchera et al. (eds.), 1985, pp.475-506); “Analysis, Results, And Future Prospective Of The TherapeuticUse Of Radiolabeled Antibody In Cancer Therapy”, in MONOCLONALANTIBODIES FOR CANCER DETECTION AND THERAPY, Baldwin et al. (eds.),1985, pp. 303-16, Academic Press; and Thorpe et al. (1982) “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates,”Immunol. Rev., 62:119-158.

An antibody or fragment thereof, with or without a therapeutic moietyconjugated to it, administered alone or in combination with cytotoxicfactor(s) and/or cytokine(s) can be used as a therapeutic.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

III. Immunizing, Screening, Identification of Antibodies andCharacterization of Monoclonal Antibodies of the Invention

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., ANTIBODIES:A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: MONOCLONAL ANTIBODIES AND T-CELLHYBRIDOMAS, pp. 563-681 (Elsevier, N.Y., 1981), both of which areincorporated by reference in their entireties. The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In anon-limiting example, mice can be immunized with an antigen of interestor a cell expressing such an antigen. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well known techniques to any suitablemyeloma cells. Hybridomas are selected and cloned by limiting dilution.The hybridoma clones are then assayed by methods known in the art forcells that secrete antibodies capable of binding the antigen. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by inoculating mice intraperitoneally with positive hybridomaclones.

In one particular embodiment, the invention provides a method forproducing monoclonal antibodies that specifically bind FcγRIIB withgreater affinity than said monoclonal antibodies bind FcγRIIAcomprising: immunizing one or more FcγRIIA transgenic mice (See U.S.Pat. No. 5,877,396 and U.S. Pat. No. 5,824,487) with the purifiedextracellular domain of human FcγRIIB, amino acids 1-180; producinghybridoma cell lines from spleen cells of said mice, screening saidhybridoma cells lines for one or more hybridoma cell lines that produceantibodies that specifically bind FcγRIIB with greater affinity thansaid antibodies bind FcγRIIA. In another specific embodiment, theinvention provides a method for producing FcγRIIB monoclonal antibodiesthat specifically bind FcγRIIB, particularly human FcγRIIB, with agreater affinity than said monoclonal antibodies bind FcγRIIA, saidmethod further comprising: immunizing one or more FcγRIIA transgenicmice with purified FcγRIIB or an immunogenic fragment thereof, boosterimmunizing said mice sufficient number of times to elicit an immuneresponse, producing hybridoma cells lines from spleen cells of said oneor more mice, screening said hybridoma cell lines for one or morehybridoma cell lines that produce antibodies that specifically bindFcγRIIB with a greater affinity than said antibodies bind FcγRIIA. Inone embodiment of the invention, said mice are immunized with purifiedFcγRIIB which has been mixed with any adjuvant known in the art toenhance immune response. Adjuvants that can be used in the methods ofthe invention include, but are not limited to, protein adjuvants;bacterial adjuvants, e.g., whole bacteria (BCG, Corynebacterium parvum,Salmonella minnesota) and bacterial components including cell wallskeleton, trehalose dimycolate, monophosphoryl lipid A, methanolextractable residue (MER) of tubercle bacillus, complete or incompleteFreund's adjuvant; viral adjuvants; chemical adjuvants, e.g., aluminumhydroxide, iodoacetate and cholesteryl hemisuccinateor; naked DNAadjuvants. Other adjuvants that can be used in the methods of theinvention include, Cholera toxin, paropox proteins, MF-59 (ChironCorporation; See also GAD65 and insulin B chain peptide (9-23) are notprimary autoantigens in the type 1 diabetes syndrome of the BB rat,which is incorporated herein by reference), MPL® (Corixa Corporation;See also Lodmell et al. (2000) “DNA Vaccination Of Mice Against RabiesVirus Effects Of The Route Of Vaccination And The AdjuvantMonophosphoryl Lipid A (MPL),” Vaccine, 18: 1059-1066; Johnson et al.(1999) “3-O-Desacyl Monophosphoryl Lipid A Derivatives: Synthesis AndImmunostimulant Activities,” Journal of Medicinal Chemistry, 42:4640-4649; Baldridge et al. (1999) “Monophosphoryl Lipid A (MPL)Formulations For The Next Generation Of Vaccines,” Methods, 19: 103-107,all of which are incorporated herein by reference), RC-529 adjuvant(Corixa Corporation; the lead compound from Corixa's aminoalkylglucosaminide 4-phosphate (AGP) chemical library, see alsowww.corixa.com), and DETOX™ adjuvant (Corixa Corporation; DETOX™adjuvant includes MPL® adjuvant (monophosphoryl lipid A) andmycobacterial cell wall skeleton; See also Eton et al. (1998) “ActiveImmunotherapy With Ultraviolet B-Irradiated Autologous Whole MelanomaCells Plus DETOX In Patients With Metastatic Melanoma,” Clin. CancerRes. 4(3):619-627; and Gupta et al. (1995) “Adjuvants For HumanVaccines—Current Status, Problems And Future Prospects,” Vaccine,13(14):1263-1276, both of which are incorporated herein by reference.)

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments may be producedby proteolytic cleavage of immunoglobulin molecules, using enzymes suchas papain (to produce Fab fragments) or pepsin (to produce F(ab′)₂fragments). F(ab′)₂ fragments contain the complete light chain, and thevariable region, the CHI region and the hinge region of the heavy chain.

For example, antibodies can also be generated using various phagedisplay methods known in the art. In phage display methods, functionalantibody domains are displayed on the surface of phage particles whichcarry the polynucleotide sequences encoding them. In a particularembodiment, such phage can be utilized to display antigen bindingdomains, such as Fab and Fv or disulfide-bond stabilized Fv, expressedfrom a repertoire or combinatorial antibody library (e.g., human ormurine). Phage expressing an antigen binding domain that binds theantigen of interest can be selected or identified with antigen, e.g.,using labeled antigen or antigen bound or captured to a solid surface orbead. Phage used in these methods are typically filamentous phage,including fd and M13. The antigen binding domains are expressed as arecombinantly fused protein to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theimmunoglobulins, or fragments thereof, of the present invention includethose disclosed in Brinkman et al. (1995) “Phage Display OfDisulfide-Stabilized Fv Fragments,” J. Immunol. Methods, 182:41-50; Ameset al. (1995) “Conversion Of Murine Fabs Isolated From A CombinatorialPhage Display Library To Full Length Immunoglobulins,” J. Immunol.Methods, 184:177-186; Kettleborough et al. (1994) “Isolation Of TumorCell-Specific Single-Chain Fv From Immunized Mice Using Phage-AntibodyLibraries And The Re-Construction Of Whole Antibodies From TheseAntibody Fragments,” Eur. J. Immunol., 24:952-958; Persic et al. (1997)“An Integrated Vector System For The Eukaryotic Expression Of AntibodiesOr Their Fragments After Selection From Phage Display Libraries,” Gene,187:9-18; Burton et al (1994) “Human Antibodies From CombinatorialLibraries,” Advances in Immunology, 57:191-280; PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired fragments, and expressed in any desired host, includingmammalian cells, insect cells, plant cells, yeast, and bacteria, e.g.,as described in detail below. For example, techniques to recombinantlyproduce Fab, Fab′ and F(ab′)₂ fragments can also be employed usingmethods known in the art such as those disclosed in PCT publication WO92/22324; Mullinax et al. (1992) “Expression Of A Heterodimeric FabAntibody Protein In One Cloning Step,” BioTechniques, 12(6):864-869; andSawai et al. (1995) “Direct Production Of The Fab Fragment Derived FromThe Sperm Immobilizing Antibody Using Polymerase Chain Reaction And cDNAExpression Vectors,” Am. J. Repr. Immunol. 34:26-34; and Better et al.(1988) “Escherichia coli Secretion Of An Active Chimeric AntibodyFragment,” Science, 240:1041-1043, each of which is incorporated byreference in its entirety). Examples of techniques which can be used toproduce single-chain Fvs and antibodies include those described in U.S.Pat. Nos. 4,946,778 and 5,258,498; Huston et al. (1991) “ProteinEngineering Of Single-Chain Fv Analogs And Fusion Proteins,” Methods inEnzymology, 203:46-88; Shu et al. (1993) “Secretion Of ASingle-Gene-Encoded Immunoglobulin From Myeloma Cells,” Proc. Nat. Acad.Sci., 90:7995-7999; and Skerra et al. (1988) “Assembly Of A FunctionalImmunoglobulin Fv Fragment In Escherichia coli,” Science, 240:1038-1040.

Phage display technology can be used to increase the affinity of anantibody of the invention for FcγRIIB. This technique would be useful inobtaining high affinity antibodies that could be used in thecombinatorial methods of the invention. The technology, referred to asaffinity maturation, employs mutagenesis or CDR walking and re-selectionusing FcγRIIB or an antigenic fragment thereof to identify antibodiesthat bind with higher affinity to the antigen when compared with theinitial or parental antibody (See, e.g., Glaser et al. (1992) “AntibodyEngineering By Codon-Based Mutagenesis In A Filamentous Phage VectorSystem,” J. Immunology 149:3903-3913). Mutagenizing entire codons ratherthan single nucleotides results in a semi-randomized repertoire of aminoacid mutations. Libraries can be constructed consisting of a pool ofvariant clones each of which differs by a single amino acid alterationin a single CDR and which contain variants representing each possibleamino acid substitution for each CDR residue. Mutants with increasedbinding affinity for the antigen can be screened by contacting theimmobilized mutants with labeled antigen. Any screening method known inthe art can be used to identify mutant antibodies with increased avidityto the antigen (e.g., ELISA) (See Wu et al. (1998) “Stepwise in vitroAffinity Maturation Of Vitaxin, An Alphav Beta3-Specific Humanized mAb,”Proc Natl. Acad. Sci. USA 95:6037-6042; Yelton et al. (1995) “AffinityMaturation Of The Br96 Anti-Carcinoma Antibody By Codon-BasedMutagenesis,” J. Immunology 155:1994-2004). CDR walking which randomizesthe light chain is also possible (See Schier et al. (1996) “Isolation OfPicomolar Affinity Anti-C-ErbB-2 Single-Chain Fv By Molecular EvolutionOf The Complementarity Determining Regions In The Center Of The AntibodyBinding Site,” J. Mol. Bio. 263:551-567.

Antibodies of the invention may be further characterized by epitopemapping, so that antibodies may be selected that have the greatestspecificity for FcγRIIB compared to FcγRIIA. Epitope mapping methods ofantibodies are well known in the art and encompassed within the methodsof the invention. In certain embodiments, fusion proteins comprising oneor more regions of FcγRIIB may be used in mapping the epitope of anantibody of the invention. In a specific embodiment, the fusion proteincontains the amino acid sequence of a region of an FcγRIIB fused to theFc portion of human IgG2. Each fusion protein may further comprise aminoacid substitutions and/or replacements of certain regions of thereceptor with the corresponding region from a homolog receptor, e.g.,FcγRIIA, as shown in Table 2 below. pMGX125 and pMGX132 contain the IgGbinding site of the FcγRIIB receptor, the former with the C-terminus ofFcγRIIB and the latter with the C-terminus of FcγRIIA and can be used todifferentiate C-terminus binding. The others have FcγRIIA substitutionsin the IgG binding site and either the FcγIIA or FcγIIB N-terminus.These molecules can help determine the part of the receptor moleculewhere the antibodies bind.

TABLE 2 Plasmid Receptor N-ter 172-180 C-ter pMGX125 RIIb IIb KKFSRSDPNAPS------SS (IIb) (SEQ ID NO: 5) (SEQ ID NO: 11) pMGX126 RIIa/b IIaQKFSRLDPN APS------SS (IIb) (SEQ ID NO: 6) (SEQ ID NO: 11) pMGX127 IIaQKFSRLDPT APS------SS (IIb) (SEQ ID NO: 7) (SEQ ID NO: 11) pMGX128 IIbKKFSRLDPT APS------SS (IIb) (SEQ ID NO: 8) (SEQ ID NO: 11) pMGX129 IIaQKFSHLDPT APS------SS (IIb) (SEQ ID NO: 9) (SEQ ID NO: 11) pMGX130 IIbKKFSHLDPT APS------SS (IIb) (SEQ ID NO: 10) (SEQ ID NO: 11) pMGX131 IIaQKFSRLDPN VPSMGSSS (IIa) (SEQ ID NO: 6) (SEQ ID NO: 12) pMGX132 IIbKKFSRSDPN VPSMGSSS (IIa) (SEQ ID NO: 5) (SEQ ID NO: 12) pMGX133RIIa-131R IIa QKFSRLDPT VPSMGSSS (IIa) (SEQ ID NO: 7) (SEQ ID NO: 12)pMGX134 RIIa-131H IIa QKFSHLDPT VPSMGSSS (IIa) (SEQ ID NO: 9) (SEQ IDNO: 12) pMGX135 IIb KKFSRLDPT VPSMGSSS (IIa) SEQ ID NO: 8) (SEQ ID NO:12) pMGX136 IIb KKFSHLDPT VPSMGSSS (IIa) (SEQ ID NO: 10) (SEQ ID NO: 12)

The fusion proteins may be used in any biochemical assay fordetermination of binding to an anti-FcγRIIB antibody of the invention,e.g., an ELISA. In other embodiments, further confirmation of theepitope specificity may be done by using peptides with specific residuesreplaced with those from the FcγRIIA sequence.

The antibodies of the invention may be characterized for specificbinding to FcγRIIB using any immunological or biochemical based methodknown in the art for characterizing including quantitating, theinteraction of the antibody to FcγRIIB. Specific binding of an antibodyof the invention to FcγRIIB may be determined for example usingimmunological or biochemical based methods including, but not limitedto, an ELISA assay, surface plasmon resonance assays,immunoprecipitation assay, affinity chromatography, and equilibriumdialysis. Immunoassays which can be used to analyze immunospecificbinding and cross-reactivity of the antibodies of the invention include,but are not limited to, competitive and non-competitive assay systemsusing techniques such as western blots, radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al., eds, 1994,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).

Antibodies of the invention may also be assayed using any surfaceplasmon resonance based assays known in the art for characterizing thekinetic parameters of the interaction of the antibody with FcγRIIB. AnySPR instrument commercially available including, but not limited to,BIAcore Instruments, available from Biacore AB (Uppsala, Sweden); IAsysinstruments available from Affinity Sensors (Franklin, Mass.); IBISsystem available from Windsor Scientific Limited (Berks, UK), SPR-CELLIAsystems available from Nippon Laser and Electronics Lab (Hokkaido,Japan), and SPR Detector Spreeta available from Texas Instruments(Dallas, Tex.) can be used in the instant invention. For a review ofSPR-based technology see Mullett et al. (2000) “Surface PlasmonResonance-Based Immunoassays,” Methods 22: 77-91; Dong et al. (2002)“Some New Aspects In Biosensors,” Reviews in Mol. Biotech., 82: 303-323;Fivash et al. (1998) “BIAcore For Macromolecular Interaction,” CurrentOpinions in Biotechnology 9: 97-101; Rich et al. (2000) “Advances InSurface Plasmon Resonance Biosensor Analysis,” Current Opinions inBiotechnology 11: 54-61; all of which are incorporated herein byreference in their entirety. Additionally, any of the SPR instrumentsand SPR based methods for measuring protein-protein interactionsdescribed in U.S. Pat. Nos. 6,373,577; 6,289,286; 5,322,798; 5,341,215;6,268,125 are contemplated in the methods of the invention, all of whichare incorporated herein by reference in their entirety.

Briefly, SPR based assays involve immobilizing a member of a bindingpair on a surface, and monitoring its interaction with the other memberof the binding pair in solution in real time. SPR is based on measuringthe change in refractive index of the solvent near the surface thatoccurs upon complex formation or dissociation. The surface onto whichthe immobilization occurs is the sensor chip, which is at the heart ofthe SPR technology; it consists of a glass surface coated with a thinlayer of gold and forms the basis for a range of specialized surfacesdesigned to optimize the binding of a molecule to the surface. A varietyof sensor chips are commercially available especially from the companieslisted supra, all of which may be used in the methods of the invention.Examples of sensor chips include those available from BIAcore AB, Inc.,e.g., Sensor Chip CM5, SA, NTA, and HPA. A molecule of the invention maybe immobilized onto the surface of a sensor chip using any of theimmobilization methods and chemistries known in the art, including butnot limited to, direct covalent coupling via amine groups, directcovalent coupling via sulfhydryl groups, biotin attachment to avidincoated surface, aldehyde coupling to carbohydrate groups, and attachmentthrough the histidine tag with NTA chips.

Antibodies of the invention may also be assayed using anyfluorescence-based assays known in the art for characterizing theinteraction of the antibody with FcγRIIB. Specific binding of anantibody of the invention to FcγRIIB may be determined, for example,using fluorescence-based methods including, but not limited to,resonance energy transer assays, anisotropy assays, quenching assays,flow cytometry assays, fluorescence correlation spectroscopy assays,two-photon excited fluorescence microscopy assays, third harmonicgeneration microscopy assays, coherent anti-stokes raman scatteringmicroscopy assays, confocal scanning microscopy assays and fluorescentimmunoassays, including, but not limited to, ELISA, etc. Such assays areroutine and well-known in the art (see, e.g., PRINCIPLES OF FLUORESCENCESPECTROSCOPY, 2nd Edition, 1999, Lakowicz (ed.); CONFOCAL SCANNINGOPTICAL MICROSCOPY AND RELATED IMAGING SYSTEMS, 1996, Corle et al.(eds.); and OPTICAL IMAGING AND MICROSCOPY: TECHNIQUES AND ADVANCEDSYSTEMS, 2003, Torok et al. (eds.), herein incorporated by reference intheir entireties).

The invention encompasses characterization of the antibodies produced bythe methods of the invention using certain characterization assays foridentifying the function of the antibodies of the invention,particularly the activity to modulate FcγRIIB signaling. For example,characterization assays of the invention can measure phosphorylation oftyrosine residues in the ITIM motif of FcγRIIB, or measure theinhibition of B cell receptor-generated calcium mobilization. Thecharacterization assays of the invention can be cell-based or cell-freeassays.

It has been well established in the art that in mast cells coaggregationof FcγRIIB with the high affinity IgE receptor, FcεRI, leads toinhibition of antigen-induced degranulation, calcium mobilization, andcytokine production (Metcalfe et al. (1997) “Mast Cells,” Physiol. Rev.77:1033-1079; Long (1999) “Regulation Of Immune Responses ThroughInhibitory Receptors,” Annu. Rev. Immunol. 17: 875-904). The moleculardetails of this signaling pathway have been recently elucidated (Ott(2002) “Downstream Of Kinase, p62(dok), Is A Mediator Of Fc gamma IIBInhibition Of Fc Epsilon RI Signaling,” J. Immunol. 162(9):4430-4439).The molecular details of this signaling pathway have been recentlyelucidated (Ott (2002) “Downstream Of Kinase, p62dok, Is A Mediator OfFcRIIB Inhibition Of FcRI Signaling,” J. Immunol. 168(9):4430-4439).Once coaggregated with FcεRI, FcγRIIB is rapidly phosphorylated ontyrosine in its ITIM motif, and then recruits Src Homology-2 containinginositol-5-phosphatase (SHIP), an SH2 domain-containing inositalpolyphosphate 5-phosphatase, which is in turn phosphorylated andassociates with Shc and p62^(dok) (p62^(dok) is the prototype of afamily of adaptor molecules, which includes signaling domains such as anaminoterminal pleckstrin homology domain (PH domain), a PTB domain, anda carboxy terminal region containing PXXP motifs and numerousphosphorylation sites (Carpino et al. (1997) “p62(dok): A ConstitutivelyTyrosine-Phosphorylated, GAP-Associated Protein In Chronic MyelogenousLeukemia Progenitor Cells,” Cell, 88: 197-204; Yamanashi et al. (1997)“Identification Of The Abl- And rasGAP-Associated 62 kDa Protein As ADocking Protein, Dok,” Cell, 88:205-211).

The invention encompasses characterizing the anti-FcγRIIB antibodies ofthe invention in modulating one or more IgE mediated responses.Preferably, cells lines co-expressing the high affinity receptor for IgEand the low affinity receptor for FcγRIIB will be used in characterizingthe anti-FcγRIIB antibodies of the invention in modulating IgE mediatedresponses. In a specific embodiment, cells from a rat basophilicleukemia cell line (RBL-H23; Barsumian et al. (1981) “IgE-InducedHistamine Release From Rat Basophilic Leukemia Cell Lines: Isolation OfReleasing And Nonreleasing Clones,” Eur. J. Immunol. 11:317-323, whichis incorporated herein by reference in its entirety) transfected withfull length human FcγRIIB will be used in the methods of the invention.RBL-2H3 is a well characterized rat cell line that has been usedextensively to study the signaling mechanisms following IgE-mediatedcell activation. When expressed in RBL-2H3 cells and coaggregated withFcεRI, FcγRIIB inhibits FcεRI-induced calcium mobilization,degranulation, and cytokine production (Malbec et al. (1998) “Fc EpsilonReceptor I-Associated Lyn-Dependent Phosphorylation Of Fc Gamma ReceptorIIB During Negative Regulation Of Mast Cell Activation,” J. Immunol.160:1647-1658; Daeron et al. (1995) “Regulation Of High-Affinity IgEReceptor-Mediated Mast Cell Activation By Murine Low-Affinity IgGReceptors,” J. Clin. Invest. 95:577-585; Ott et al. (2002) “DownstreamOf Kinase, p62(dok), Is A Mediator Of Fc gamma IIB Inhibition Of FcEpsilon RI Signaling,” J. Immunol. 168:4430-4439).

In some embodiments, the invention encompasses characterizing theanti-FcγRIIB antibodies of the invention for inhibition of FcεRI inducedmast cell activation. For example, cells from a rat basophilic leukemiacell line (RBL-H23; Barsumian et al. (1981) “IgE-Induced HistamineRelease From Rat Basophilic Leukemia Cell Lines: Isolation Of ReleasingAnd Nonreleasing Clones,” Eur. J. Immunol. 11:317-323) that have beentransfected with FcγRIIB are sensitized with IgE and stimulated eitherwith F(ab′)₂ fragments of rabbit anti-mouse IgG, to aggregate FcεRIalone, or with whole rabbit anti-mouse IgG to coaggregate FcγRIIB andFcεRI. In this system, indirect modulation of down stream signalingmolecules can be assayed upon addition of antibodies of the invention tothe sensitized and stimulated cells. For example, tyrosinephosphorylation of FcγRIIB and recruitment and phosphorylation of SHIP,activation of MAP kinase family members, including but not limited toErk1, Erk2, JNK, or p38; and tyrosine phosphorylation of p62^(dok) andits association with SHIP and RasGAP can be assayed.

One exemplary assay for determining the inhibition of FcεRI induced mastcell activation by the antibodies of the invention can comprise of thefollowing: transfecting RBL-H23 cells with human FcγRIIB; sensitizingthe RBL-H23 cells with IgE; stimulating RBL-H23 cells with eitherF(ab′)₂ of rabbit anti-mouse IgG (to aggregate FcεRI alone and elicitFcεRI-mediated signaling, as a control), or stimulating RBL-H23 cellswith whole rabbit anti-mouse IgG to (to coaggregate FcγRIIB and FcεRI,resulting in inhibition of FcεRI-mediated signaling). Cells that havebeen stimulated with whole rabbit anti-mouse IgG antibodies can befurther pre-incubated with the antibodies of the invention. MeasuringFcεRI-dependent activity of cells that have been pre-incubated with theantibodies of the invention and cells that have not been pre-incubatedwith the antibodies of the invention, and comparing levels ofFcεRI-dependent activity in these cells, would indicate a modulation ofFcεRI-dependent activity by the antibodies of the invention.

The exemplary assay described above can be for example, used to identifyantibodies that block ligand (IgG) binding to FcγRIIB receptor andantagonize FcγRIIB-mediated inhibition of FcεRI signaling by preventingcoaggregating of FcγRIIB and FcεRI. This assay likewise identifiesantibodies that enhance coaggregation of FcγRIIB and FcεRI and agonizeFcγRIIB-mediated inhibition of FcεRI signaling by promotingcoaggregating of FcγRIIB and FcεRI.

In a preferred embodiment, FcεRI-dependent activity is at least one ormore of the following: modulation of downstream signaling molecules(e.g., modulation of phosphorylation state of FcγRIIB, modulation ofSHIP recruitment, modulation of MAP Kinase activity, modulation ofphosphorylation state of SHIP, modulation of SHIP and Shc associationSHIP and Shc, modulation of the phosphorylation state of p62^(dok),modulation of p62^(dok) and SHIP association, modulation of p62^(dok)and RasGAP association, modulation of calcium mobilization, modulationof degranulation, and modulation of cytokine production. In yet anotherpreferred embodiment, FcεRI-dependent activity is serotonin releaseand/or extracellular Ca⁺⁺ influx and/or IgE dependent mast cellactivation. It is known to one skilled in the art that coaggregation ofFcγRIIB and FcεRI stimulates FcγRIIB tyrosine phosphorylation,stimulates recruitment of SHIP, stimulates SHIP tyrosine phosphorylationand association with Shc, and inhibits activation of MAP kinase familymembers including, but not limited to, Erk1, Erk2, JNK, p38. It is alsoknown to those skilled in the art that coaggregation of FcγRIIB andFcεRI stimulates enhanced tyrosine phosphorylation of p62^(dok) and itsassociation with SHIP and RasGAP.

In some embodiments, the anti-FcγRIIB antibodies of the invention arecharacterized for their ability to modulate an IgE mediated response bymonitoring and/or measuring degranulation of mast cells or basophils,preferably in a cell-based assay. Preferably, mast cells or basophilsfor use in such assays have been engineered to contain human FcγRIIBusing standard recombinant methods known to one skilled in the art. In aspecific embodiment the anti-FcγRIIB antibodies of the invention arecharacterized for their ability to modulate an IgE mediated response ina cell-based β-hexosaminidase (enzyme contained in the granules) releaseassay. β-hexosaminidase release from mast cells and basophils is aprimary event in acute allergic and inflammatory condition (Aketani etal. (2001) “Correlation Between Cytosolic Calcium Concentration AndDegranulation In RBL-2H3Cells In The Presence Of Various ConcentrationsOf Antigen-Specific IgEs,” Immunol. Lett. 75: 185-189; Aketani et al.(2000) “A Screening Method For Antigen-Specific IgE Using Mast CellsBased On Intracellular Calcium Signaling,” Anal. Chem. 72: 2653-2658).Release of other inflammatory mediators including but not limited toserotonin and histamine may be assayed to measure an IgE mediatedresponse in accordance with the methods of the invention. Although notintending to be bound by a particular mechanism of action, release ofgranules such as those containing β-hexosaminidase from mast cells andbasophils is an intracellular calcium concentration dependent processthat is initiated by the cross-linking of FcεRIs with multivalentantigen.

One exemplary assay for characterizing the anti-FcγRIIB antibodies ofthe invention in mediating an IgE mediated response is aβ-hexosaminidase release assay comprising the following: transfectingRBL-H23 cells with human FcγRIIB; sensitizing the cells with mouse IgEalone or with mouse IgE and an anti-FcγRIIB antibody of the invention;stimulating the cells with various concentrations of goat anti-mouseF(ab)₂, preferably in a range from 0.03 μg/mL to 30 μg/mL for about 1hour; collecting the supernatant; lysing the cells; and measuring theβ-hexosaminidase activity released in the supernatant by a colorometricassay, e.g., using p-nitrophenyl N-acetyl-β-D-glucosaminide. Thereleased β-hexosaminidase activity is expressed as a percentage of thereleased activity to the total activity. The released β-hexosaminidaseactivity will be measured and compared in cells treated with antigenalone; IgE alone; IgE and an anti-FcγRIIB antibody of the invention.Although not intending to be bound by a particular mechanism of action,once cells are sensitized with mouse IgE alone and challenged withF(ab)₂ fragments of a polyclonal goat anti-mouse IgG, aggregation andcross linking of FcεRI occurs since the polyclonal antibody recognizesthe light chain of the murine IgE bound to the FcεRI; which in turnleads to mast cell activation and degranulation. On the other hand, whencells are sensitized with mouse IgE and an anti-FcγRIIB antibody of theinvention and challenged with F(ab)₂ fragments of a polyclonal goatanti-mouse IgG; cross linking of FcεRI and FcγRIIB occurs, resulting ininhibition of FcεRI induced degranulation. In either case, goat antimouse F(ab)₂ induces a dose-dependent β-hexoaminidase release. In someembodiments, the anti-FcγRIIB antibodies bound to the FcγRIIB receptorand cross linked to FcεRI do not affect the activation of the inhibitorypathway, i.e., there is no alteration in the level of degranulation inthe presence of an anti-FcγRIIB antibody. In other embodiments, theanti-FcγRIIB antibodies mediate a stronger activation of the inhibitoryreceptor, FcγRIIB, when bound by the anti-FcγRIIB antibody, allowingeffective cross linking to FcεRI and activation of the inhibitorypathway of homo-aggregated FcγRIIB.

The invention also encompasses characterizing the effect of theanti-FcγRIIB antibodies of the invention on IgE mediated cell responseusing calcium mobilization assays using methodologies known to oneskilled in the art. An exemplary calcium mobilization assay may comprisethe following: priming basophils or mast cells with IgE; incubating thecells with a calcium indicator, e.g., Fura 2; stimulating cells asdescribed supra; and monitoring and/or quantitating intracellularcalcium concentration for example by using flow cytometry. The inventionencompasses monitoring and/or quantitating intracellular calciumconcentration by any method known to one skilled in the art see, e.g.,Aketani et al. (2001) “Correlation Between Cytosolic CalciumConcentration And Degranulation In RBL-2H3Cells In The Presence OfVarious Concentrations Of Antigen-Specific IgEs,” Immunology Letters75:185-189; Oka et al. (2002) “FcRI Cross-Linking-Induced Actin AssemblyMediates Calcium Signalling In RBL-2H3 Mast Cells,” British J. of Pharm.136:837-845; Ott et al. (2002) “Downstream Of Kinase, p62dok, Is AMediator Of FcRIIB Inhibition Of FcRI Signaling,” J. of Immunology,168:4430-4439 and Mahmoud et al. (2001) “Microdomains Of High CalciumAre Not Required For Exocytosis In RBL-2H3 Mucosal Mast Cells,” J. CellBiol., 153(2):339-350; all of which are incorporated herein byreference.

In preferred embodiments, anti-FcγRIIB antibodies of the inventioninhibit IgE mediated cell activation. In other embodiments, theanti-FcγRIIB antibodies of the invention block the inhibitory pathwaysregulated by FcγRIIB or block the ligand binding site on FcγRIIB andthus enhance immune response.

The ability to study human mast cells has been limited by the absence ofsuitable long term human mast cell cultures. Recently two novel stemcell factor dependent human mast cell lines, designated LAD 1 and LAD2,were established from bone marrow aspirates from a patient with mastcell sarcoma/leukemia (Kirshenbaum et al. (2003) “Characterization OfNovel Stem Cell Factor Responsive Human Mast Cell Lines LAD 1 And 2Established From A Patient With Mast Cell Sarcoma/Leukemia; ActivationFollowing Aggregation Of FcepsilonRI Or FcgammaRI,” Leukemia research,27(8):677-682, which is incorporated herein by reference in itsentirety). Both cell lines have been described to express FcεRI andseveral human mast cell markers. The invention encompasses using LAD 1and 2 cells in the methods of the invention for assessing the effect ofthe antibodies of the invention on IgE mediated responses. In a specificembodiment, cell-based β-hexosaminidase release assays such as thosedescribed supra may be used in LAD cells to determine any modulation ofthe IgE-mediated response by the anti-FcγRIIB antibodies of theinvention. In an exemplary assay, human mast cells, e.g., LAD 1, areprimed with chimaeric human IgE anti-nitrophenol (NP) and challengedwith BSA-NP, the polyvalent antigen, and cell degranulation is monitoredby measuring the β-hexosaminidase released in the supernatant(Kirshenbaum et al. (2003) “Characterization Of Novel Stem Cell FactorResponsive Human Mast Cell Lines LAD 1 And 2 Established From A PatientWith Mast Cell Sarcoma/Leukemia; Activation Following Aggregation OfFcepsilonRI Or FcgammaRI,” Leukemia research, 27(8):677-682, which isincorporated herein by reference in its entirety).

In some embodiments, if human mast cells have a low expression ofendogenous FcγRIIB, as determined using standard methods known in theart, e.g., FACS staining, it may be difficult to monitor and/or detectdifferences in the activation of the inhibitory pathway mediated by theanti-FcγRIIB antibodies of the invention. The invention thus encompassesalternative methods, whereby the FcγRIIB expression may be upregulatedusing cytokines and particular growth conditions. FcγRIIB has beendescribed to be highly up-regulated in human monocyte cell lines, e.g.,THP1 and U937, (Tridandapani et al. (2002) “Regulated Expression AndInhibitory Function Of FcRIIb In Human Monocytic Cells,” J. Biol. Chem.,277(7): 5082-5089) and in primary human monocytes (Pricop et al. (2001)“Differential Modulation Of Stimulatory And Inhibitory Fc GammaReceptors On Human Monocytes By Th1 And Th2 Cytokines,” J. Immunol.,166: 531-537). Differentiation of U937 cells with dibutyryl cyclic AMPhas been described to increase expression of FcγRII (Cameron et al.(2002) “Differentiation Of The Human Monocyte Cell Line, U937, WithDibutyryl CyclicAMP Induces The Expression Of The Inhibitory FcReceptor, FcgammaRIIB,” Immunology Letters 83, 171-179). Thus theendogenous FcγRIIB expression in human mast cells for use in the methodsof the invention may be up-regulated using cytokines, e.g., IL-4, IL-13,in order to enhance sensitivity of detection.

The invention also encompasses characterizing the anti-FcγRIIBantibodies of the invention for inhibition of B-cell receptor(BCR)-mediated signaling. BCR-mediated signaling can include at leastone or more down stream biological responses, such as activation andproliferation of B cells, antibody production, etc. Coaggregation ofFcγRIIB and BCR leads to inhibition of cell cycle progression andcellular survival. Further, coaggregation of FcγRIIB and BCR leads toinhibition of BCR-mediated signaling.

Specifically, BCR-mediated signaling comprises at least one or more ofthe following: modulation of down stream signaling molecules (e.g.,phosphorylation state of FcγRIIB, SHIP recruitment, localization of Btkand/or PLCγ, MAP kinase activity, recruitment of Akt (anti-apoptoticsignal), calcium mobilization, cell cycle progression, and cellproliferation.

Although numerous effector functions of FcγRIIB-mediated inhibition ofBCR signaling are mediated through SHIP, recently it has beendemonstrated that lipopolysaccharide (LPS)-activated B cells from SHIPdeficient mice exhibit significant FcγRIIB-mediated inhibition ofcalcium mobilization, Ins(1,4,5)P₃ production, and Erk and Aktphosphorylation (Brauweiler et al. (2001) “Partially Distinct MolecularMechanisms Mediate Inhibitory FcgammaRIIB Signaling In Resting AndActivated B Cells,” J. Immunol. 167(1): 204-211). Accordingly, ex vivo Bcells from SHIP deficient mice can be used to characterize theantibodies of the invention. One exemplary assay for determiningFcγRIIB-mediated inhibition of BCR signaling by the antibodies of theinvention can comprise the following: isolating splenic B cells fromSHIP deficient mice, activating said cells with lipopolysachharide, andstimulating said cells with either F(ab′)₂ anti-IgM to aggregate BCR orwith anti-IgM to coaagregate BCR with FcγRIIB. Cells that have beenstimulated with intact anti-IgM to coaggregate BCR with FcγRIIB can befurther pre-incubated with the antibodies of the invention.FcγRIIB-dependent activity of cells can be measured by standardtechniques known in the art. Comparing the level of FcγRIIB-dependentactivity in cells that have been pre-incubated with the antibodies ofthe invention and cells that have not been pre-incubated, and comparingthe levels would indicate a modulation of FcγRIIB-dependent activity bythe antibodies of the invention.

Measuring FcγRIIB-dependent activity can include, for example, measuringintracellular calcium mobilization by flow cytometry, measuringphosphorylation of Akt and/or Erk, measuring BCR-mediated accumulationof PI(3,4,5)P₃, or measuring FcγRIIB-mediated proliferation B cells.

The assays can be used, for example, to identify antibodies thatmodulate FcγRIIB-mediated inhibition of BCR signaling by blocking theligand (IgG) binding site to FcγRIIB receptor and antagonizingFcγRIIB-mediated inhibition of BCR signaling by preventing coaggregationof FcγRIIB and BCR. The assays can also be used to identify antibodiesthat enhance coaggregation of FcγRIIB and BCR and agonizeFcγRIIB-mediated inhibition of BCR signaling.

The invention relates to characterizing the anti-FcγRIIB antibodies ofthe invention for FcγRII-mediated signaling in humanmonocytes/macrophages. Coaggregation of FcγRIIB with a receptor bearingthe immunoreceptor tyrosine-based activation motif (ITAM) acts todown-regulate FcγR-mediated phagocytosis using SHIP as its effector(Tridandapani et al. (2002) “Regulated Expression And InhibitoryFunction Of FcRIIb In Human Monocytic Cells,” J. Biol. Chem.277(7):5082-5089). Coaggregation of FcγRIIA with FcγRIIB results inrapid phosphorylation of the tyrosine residue on FcγRIIB's ITIM motif,leading to an enhancement in phosphorylation of SHIP, association ofSHIP with Shc, and phosphorylation of proteins having the molecularweight of 120 and 60-65 kDa. In addition, coaggregation of FcγRIIA withFcγRIIB results in down-regulation of phosphorylation of Akt, which is aserine-threonine kinase that is involved in cellular regulation andserves to suppress apoptosis.

The invention further encompasses characterizing the anti-FcγRIIBantibodies of the invention for their inhibition of FcγR-mediatedphagocytosis in human monocytes/macrophages. For example, cells from ahuman monocytic cell line, THP-1 can be stimulated either with Fabfragments of mouse monoclonal antibody IV.3 against FcγRII and goatanti-mouse antibody (to aggregate FcγRIIA alone), or with whole IV.3mouse monoclonal antibody and goat anti-mouse antibody (to coaggregateFcγRIIA and FcγRIIB). In this system, modulation of down streamsignaling molecules, such as tyrosine phosphorylation of FcγRIIB,phosphorylation of SHIP, association of SHIP with Shc, phosphorylationof Akt, and phosphorylation of proteins having the molecular weight of120 and 60-65 kDa can be assayed upon addition of antibodies of theinvention to the stimulated cells. In addition, FcγRIIB-dependentphagocytic efficiency of the monocyte cell line can be directly measuredin the presence and absence of the antibodies of the invention.

Another exemplary assay for determining inhibition of FcγR-mediatedphagocytosis in human monocytes/macrophages by the antibodies of theinvention can comprise the following: stimulating THP-1 cells witheither Fab of IV.3 mouse anti-FcγRII antibody and goat anti-mouseantibody (to aggregate FcγRIIA alone and elicit FcγRIIA-mediatedsignaling); or with mouse anti-FcγRII antibody and goat anti-mouseantibody (to coaggregate FcγRIIA and FcγRIIB and inhibitingFcγRIIA-mediated signaling. Cells that have been stimulated with mouseanti-FcγRII antibody and goat anti-mouse antibody can be furtherpre-incubated with the antibodies of the invention. MeasuringFcγRIIA-dependent activity of stimulated cells that have beenpre-incubated with antibodies of the invention and cells that have notbeen pre-incubated with the antibodies of the invention and comparinglevels of FcγRIIA-dependent activity in these cells would indicate amodulation of FcγRIIA-dependent activity by the antibodies of theinvention.

The exemplary assay described can be used for example, to identifyantibodies that block ligand binding of FcγRIIB receptor and antagonizeFcγRIIB-mediated inhibition of FcγRIIA signaling by preventingcoaggregation of FcγRIIB and FcγRIIA. This assay likewise identifiesantibodies that enhance coaggregation of FcγRIIB and FcγRIIA and agonizeFcγRIIB-mediated inhibition of FcγRIIA signaling.

In another embodiment of the invention, the invention relates tocharacterizing the function of the antibodies of the invention bymeasuring the ability of THP-1 cells to phagocytose fluoresceinatedIgG-opsonized sheep red blood cells (SRBC) by methods previouslydescribed (Tridandapani et al. (2000) “The Adapter Protein LAT EnhancesFc Receptor-Mediated Signal Transduction In Myeloid Cells,” J. Biol.Chem. 275: 20480-20487). For example, an exemplary assay for measuringphagocytosis comprises of: treating THP-1 cells with the antibodies ofthe invention or with a control antibody that does not bind to FcγRII,comparing the activity levels of said cells, wherein a difference in theactivities of the cells (e.g., rosetting activity (the number of THP-1cells binding IgG-coated SRBC), adherence activity (the total number ofSRBC bound to THP-1 cells), and phagocytic rate) would indicate amodulation of FcγRIIA-dependent activity by the antibodies of theinvention. This assay can be used to identify, for example, antibodiesthat block ligand binding of FcγRIIB receptor and antagonizeFcγRIIB-mediated inhibition of phagocytosis. This assay can alsoidentify antibodies that enhance FcγRIIB-mediated inhibition of FcγRIIAsignaling.

In a preferred embodiment, the antibodies of the invention modulateFcγRIIB-dependent activity in human monocytes/macrophages in at leastone or more of the following ways: modulation of downstream signalingmolecules (e.g., modulation of phosphorylation state of FcγRIIB,modulation of SHIP phosphorylation, modulation of SHIP and Shcassociation, modulation of phosphorylation of Akt, modulation ofphosphorylation of additional proteins around 120 and 60-65 kDa) andmodulation of phagocytosis.

The invention encompasses characterization of the antibodies of theinvention using assays known to those skilled in the art for identifyingthe effect of the antibodies on effector cell function of therapeuticantibodies, e.g., their ability to enhance tumor-specific ADCC activityof therapeutic antibodies. Therapeutic antibodies that may be used inaccordance with the methods of the invention include but are not limitedto anti-tumor antibodies, anti-viral antibodies, anti-microbialantibodies (e.g., bacterial and unicellular parasites), examples ofwhich are disclosed herein. In particular, the invention encompassescharacterizing the antibodies of the invention for their effect onFcγR-mediated effector cell function of therapeutic antibodies, e.g.,tumor-specific monoclonal antibodies. Examples of effector cellfunctions that can be assayed in accordance with the invention, includebut are not limited to, antibody-dependent cell mediated cytotoxicity,phagocytosis, opsonization, opsonophagocytosis, C1q binding, andcomplement dependent cell mediated cytotoxicity. Any cell-based or cellfree assay known to those skilled in the art for determining effectorcell function activity can be used (For effector cell assays, seePerussia et al. (2000) “Assays For Antibody-Dependent Cell-MediatedCytotoxicity (ADCC) And Reverse ADCC (redirected cytotoxicity) In HumanNatural Killer Cells,” Methods Mol. Biol. 121: 179-192; Lehmann et al.(2000) “Phagocytosis: Measurement By Flow Cytometry,” J. Immunol.Methods, 243(1-2): 229-242; Brown (1994) “In Vitro Assays Of PhagocyticFunction Of Human Peripheral Blood Leukocytes: Receptor Modulation AndSignal Transduction,” Methods Cell Biol., 45: 147-164; Munn et al.(1990) “Phagocytosis Of Tumor Cells By Human Monocytes Cultured InRecombinant Macrophage Colony-Stimulating Factor,” J. Exp. Med., 172:231-237, Abdul-Majid et al. (2002) “Fc Receptors Are Critical ForAutoimmune Inflammatory Damage To The Central Nervous System InExperimental Autoimmune Encephalomyelitis,” Scand. J. Immunol. 55:70-81; Ding et al. (1998) “Two Human T Cell Receptors Bind In A SimilarDiagonal Mode To The HLA-A2/Tax Peptide Complex Using Different TCRAmino Acids,” Immunity 8:403-411, each of which is incorporated byreference herein in its entirety).

Antibodies of the invention can be assayed for their effect onFcγR-mediated ADCC activity of therapeutic antibodies in effector cells,e.g., natural killer cells, using any of the standard methods known tothose skilled in the art (See e.g., Perussia et al. (2000) “Assays ForAntibody-Dependent Cell-Mediated Cytotoxicity (ADCC) And Reverse ADCC(redirected cytotoxicity) In Human Natural Killer Cells,” Methods Mol.Biol. 121: 179-192). “Antibody-dependent cell-mediated cytotoxicity” and“ADCC” as used herein carry their ordinary and customary meaning in theart and refer to an in vitro cell-mediated reaction in which nonspecificcytotoxic cells that express FcγRs (e.g., monocytic cells such asNatural Killer (NK) cells and macrophages) recognize bound antibody on atarget cell and subsequently cause lysis of the target cell. Inprinciple, any effector cell with an activating FcγR can be triggered tomediate ADCC. The primary cells for mediating ADCC are NK cells whichexpress only FcγRIII, whereas monocytes, depending on their state ofactivation, localization, or differentiation, can express FcγRI, FcγRII,and FcγRIII. For a review of FcγR expression on hematopoietic cells see,e.g., Ravetch et al. (1991) “Fc Receptors,” Annu. Rev. Immunol.,9:457-492, which is incorporated herein by reference in its entirety.

Effector cells are leukocytes which express one or more FcγRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Effector cells that may beused in the methods of the invention include but are not limited toperipheral blood mononuclear cells (PBMC), natural killer (NK) cells,monocytes, and neutrophils; with PBMCs and NK cells being preferred. Theeffector cells may be isolated from a native source thereof, e.g., fromblood or PBMCs as described herein. Preferably, the effector cells usedin the ADCC assays of the invention are peripheral blood mononuclearcells (PBMC) that are preferably purified from normal human blood, usingstandard methods known to one skilled in the art, e.g., usingFicoll-Paque density gradient centrifugation. For example, PBMCs may beisolated by layering whole blood onto Ficoll-Hypaque and spinning thecells at 500 g, at room temperature for 30 minutes. The leukocyte layercan be harvested as effector cells. Other effector cells that may beused in the ADCC assays of the invention include but are not limited tomonocyte-derived macrophages (MDMs). MDMs that are used as effectorcells in the methods of the invention, are preferably obtained as frozenstocks or used fresh, (e.g., from Advanced Biotechnologies, MD). In mostpreferred embodiments, elutriated human monocytes are used as effectorcells in the methods of the invention. Elutriated human monocytesexpress activating receptors, FcγRIIIA and FcγRIIA and the inhibitoryreceptor, FcγRIIB. Human monocytes are commercially available and may beobtained as frozen stocks, thawed in basal medium containing 10% humanAB serum or in basal medium with human serum containing cytokines.Levels of expression of FcγRs in the cells may be directly determined;e.g. using FACS analysis. Alternatively, cells may also be allowed tomature to macrophages in culture. The level of FcγRIIB expression may beincreased in macrophages. Antibodies that may be used in determining theexpression level of FcγRs include but are not limited to anti-humanFcγRIIA antibodies, e.g., IV.3-FITC; anti-FcγRI antibodies, e.g., 32.2FITC; and anti-FcγRIIIA antibodies, e.g., 3G8-PE.

Target cells used in the ADCC assays of the invention include, but arenot limited to, breast cancer cell lines, e.g., SK-BR-3 with ATCCaccession number HTB-30 (see, e.g., Tremp et al. (1976) “Human BreastCancer In Culture,” Recent Results Cancer Res. 57:33-41); B-lymphocytes;cells derived from Burkitts lymphoma, e.g., Raji cells with ATCCaccession number CCL-86 (see, e.g., Epstein et al. (1965)“Characteristics And Mode Of Growth Of Tissue Culture Strain (EB1) OfHuman Lymphoblasts From Burkitt's Lymphoma,” J. Natl. Cancer Inst. 34:231-240), Daudi cells with ATCC accession number CCL-213 (see, e.g.,Klein et al. (1968) “Surface IgM-Kappa Specificity On A Burkitt LymphomaCell In Vivo And In Derived Culture Lines,” Cancer Res. 28: 1300-1310);ovarian carcinoma cell lines, e.g., OVCAR-3 with ATCC accession numberHTB-161 (see, e.g., Hamilton et al. (1983) “Characterization Of A HumanOvarian Carcinoma Cell Line (NIH:OVCAR-3) With Androgen And EstrogenReceptors,” Cancer Res. 43(11):5379-5389), SK-OV-3, PA-1, CAOV3, OV-90,and IGROV-1 (available from the NCl repository; Benard et al. (1985)“Characterization Of A Human Ovarian Adenocarcinoma Line, IGROV1, InTissue Culture And In Nude Mice,” Cancer Research, 45:4970-4979; whichis incorporated herein by reference in its entirety. The target cellsmust be recognized by the antigen binding site of the antibody to beassayed. The target cells for use in the methods of the invention mayhave low, medium, or high expression level of a cancer antigen. Theexpression levels of the cancer antigen may be determined using commonmethods known to one skilled in the art, e.g., FACS analysis. Forexample, the invention encompasses the use of ovarian cancer cells suchas IGROV-1, wherein Her2/neu is expressed at different levels, orOV-CAR-3 (ATCC Assession Number HTB-161; characterized by a lowerexpression of Her2/neu than SK-BR-3, the breast carcinoma cell line).Other ovarian carcinoma cell lines that may be used as target cells inthe methods of the invention include OVCAR-8 (Hamilton et al. (1983)“Characterization Of A Human Ovarian Carcinoma Cell Line (NIH: OVCAR-3)With Androgen And Estrogen Receptors,” Cancer Res. 43(11):5379-5389);SK-OV-3 (ATCC Accession Number HTB-77); Caov-3 (ATCC Accession NumberHTB-75); PA-1 (ATCC Accession Number CRL-1572); OV-90 (ATCC AccessionNumber CRL-11732); and OVCAR-4. Other breast cancer cell lines that maybe used in the methods of the invention include BT-549 (ATCC AccessionNumber HTB-122), MCF7 (ATCC Accession Number HTB-22), and Hs578T (ATCCAccession Number HTB-126), all of which are available from the NClrepository and ATCC and incorporated herein by reference. Other celllines that may be used in the methods of the invention include but arenot limited to CCRF-CEM (leukemia); HL-60 (TB, leukemia); MOLT-4(leukemia); RPMI-8226 (leukemia); SR (leukemia); A549 (Non-small celllung); EKVX (Non-small cell lung); HOP-62 (Non-small cell lung); HOP-92(Non-small cell lung); NC1-H226 (Non-small cell lung); NC1-H23(Non-small cell lung); NC1-H322M (Non-small cell lung); NC1-H460(Non-small cell lung); NC1-H522 (Non-small cell lung); COLO 205 (Colon);HCC-2998 (Colon); HCT-116 (Colon); HCT-15 (Colon); HT29 (Colon); KM12(Colon); SW-620 (Colon); SF-268 (CNS); SF-295 (CNS); SF-539 (CNS);SNB-19 (CNS); SNB-75 (CNS); U251 (CNS); LOX 1MV1 (Melanoma); MALME-3M(Melanoma); M14 (Melanoma); SK-MEL-2 (Melanoma); SK-MEL-28 (Melanoma);SK-MEL-5 (Melanoma); UACC-257 (Melanoma); UACC-62 (Melanoma); IGR-OV1(Ovarian); OVCAR-3, 4, 5, 8 (Ovarian); SK-OV-3 (Ovarian); 786-0 (Renal);A498 (Renal); ACHN (Renal); CAK1-1 (Renal); SN12C (Renal); TK-10(Renal); UO-31 (Renal); PC-3C (Prostate); DU-145 (Prostate); NCl/ADR-RES(Breast); MDA-MB-231/ATCC (Breast); MDA-MB-435 (Breast); DMS 114(Small-cell lung); and SHP-77 (Small-cell lung); all of which areavailable from the NCl and incorporated herein by reference.

An exemplary assay for determining the effect of the antibodies of theinvention on the ADCC activity of therapeutic antibodies is based on a⁵¹Cr release assay comprising of: labeling target cells with[⁵¹Cr]Na₂CrO₄ (this cell-membrane permeable molecule is commonly usedfor labeling since it binds cytoplasmic proteins and althoughspontaneously released from the cells with slow kinetics, it is releasedmassively following target cell lysis); preferably, the target cellsexpress one or more tumor antigens, osponizing the target cells with oneor more antibodies that immunospecifically bind the tumor antigensexpressed on the cell surface of the target cells, in the presence andabsence of an antibody of the invention, e.g., 2B6, 3H7, combining theopsonized radiolabeled target cells with effector cells in a microtitreplate at an appropriate ratio of target cells to effector cells;incubating the mixture of cells preferably for 16-18 hours, preferablyat 37° C.; collecting supernatants; and analyzing the radioactivity inthe supernatant samples. The cytotoxicity of the therapeutic antibodiesin the presence and absence of the antibodies of the invention can thenbe determined, for example using the following formula: Percent specificlysis=(Experimental lysis-antibody-independent lysis/maximallysis−antibody independent lysis)×100%. A graph can be generated byvarying either the target:effector cell ratio or antibody concentration.

In yet another embodiment, the antibodies of the invention arecharacterized for antibody dependent cellular cytotoxicity (ADCC) inaccordance with the method described earlier, see, e.g., Ding et al.(1998) “Two Human T Cell Receptors Bind In A Similar Diagonal Mode ToThe HLA-A2/Tax Peptide Complex Using Different TCR Amino Acids,”Immunity 8:403-411; which is incorporated herein by reference in itsentirety.

In some embodiments, the invention encompasses characterizing thefunction of the antibodies of the invention in enhancing ADCC activityof therapeutic antibodies in an in vitro based assay and/or in an animalmodel.

In a specific embodiment, the invention encompasses determining thefunction of the antibodies of the invention in enhancing tumor specificADCC using an ovarian cancer model and/or breast cancer model.

Preferably, the ADCC assays of the invention are done using more thanone cancer cell line, characterized by the expression of at least onecancer antigen, wherein the expression level of the cancer antigen isvaried among the cancer cell lines used. Although not intending to bebound by a particular mechanism of action, performing ADCC assays inmore than one cell line wherein the expression level of the cancerantigen is varied, will allow determination of stringency of tumorclearance of the antibodies of the invention. In one embodiment, theADCC assays of the invention are done using cancer cell lines withdifferent levels of expression of a cancer antigen.

In an exemplary assay, OVCAR3, an ovarian carcinoma cell line can serveas the tumor target expressing the tumor antigens, Her2/neu and TAG-72;human monocytes, that express the activating FcγRIIIA and FcγRIIA andinhibitory FcγRIIB, can be used as effectors; and tumor specific murineantibodies, ch4D5 and chCC49, can be used as the tumor specificantibodies. OVCAR-3 cells are available from ATCC (Accession NumberHTB-161). Preferably, OVCAR-3 cells are propagated in mediumsupplemented with 0.01 mg/ml bovine insulin. 5×10⁶ viable OVCAR-3 cellsmay be injected subcutaneously (s.c) into age and weight matched nudeathymic mice with Matrigel (Becton Dickinson). The estimated weight ofthe tumor can be calculated by the formula: length-(width)²/2, andpreferably does not exceed 3 grams. Anchorage-dependent tumor can beisolated after 6-8 weeks, and the cells can be dissociated by adding 1μg of Collagenase (Sigma) per gram of tumor and a 5 mg/mL RNase, passedthrough a cell strainer and nylon mesh to isolate cells. Cells can thenbe frozen for long-term storage for s.c. injection for establishment ofthe xenograft model.

Hybridomas secreting CC49 and 4D5 antibodies are available with ATCCAccession Numbers HB-9459 and CRL-3D463 and the heavy chain and lightchain nucleotide sequences are in the public domain (Murray et al.(1994) “Phase II Radioimmunotherapy Trial With 131I-CC49 In ColorectalCancer,” Cancer 73 (35):1057-1066, Yamamoto et al. (1986) “Similarity OfProtein Encoded By The Human c-erb-B-2 Gene To Epidermal Growth FactorReceptor,” Nature, 319:230-234; both of which are incorporated herein byreference in their entirety), Preferably, the 4D5 and CC49 antibodiesare chimerized using standard methods known to one skilled in the art sothat the human Fc sequence, e.g., human constant region of IgG1, isgrafted onto the variable region of the murine antibodies in order toprovide the effector function. The chimeric 4D5 and CC49 antibodies bindvia their variable region to the target cell lines and via their Fcregion to FcγRs expressed on human effector cells. CC49 is directed toTAG-72; a high molecular weight mucin that is highly expressed on manyadenocarcinoma cells and ovarian carcinoma (Lottich et al. (1985)“Tumor-Associated Antigen TAG-72: Correlation Of Expression In PrimaryAnd Metastatic Breast Carcinoma Lesions,” Breast Cancer Res. Treat.6(1):49-56; Mansi et al. (1989) “Diagnosis Of Ovarian Cancer WithRadiolabelled Monoclonal Antibodies: Our Experience Using 131I-B72.3,”Int. J. Rad. Appl. Instrum B. 16(2):127-135; Colcher et al. (1991) “Invivo And In Vitro Clinical Applications Of Monoclonal Antibodies AgainstTAG-72,” Int. J. Rad. Appl. Instrum B. 18:395-441; all of which areincorporated herein by reference in their entirety). 4D5 is directed tohuman epidermal growth factor receptor 2 (Carter et al. (1992)“Humanization Of An Anti-p185HER2Antibody For Human Cancer Therapy,”Proc. Natl. Acad. Sci. USA, 89: 4285-4289 which is incorporated hereinby reference in its entirety). Antibodies of the invention can then beutilized to investigate the enhancement of ADCC activity of the tumorspecific antibodies, by blocking the inhibitory FcγRIIB. Although notintending to be bound by a particular mechanism of action, uponactivation of effector cells that express at least one activating FcγR,e.g., FcγRIIA, the expression of the inhibitory receptor (FcγRIIB) isenhanced and this limits the clearance of tumors as the ADCC activity ofFcγRIIA is suppressed. However, antibodies of the invention can serve asa blocking antibody, i.e., an antibody that will prevent the inhibitorysignal from being activated and thus the activation signal, e.g., ADCCactivity, will be sustained for a longer period and may result in potenttumor clearance.

Preferably, the antibodies of the invention for use in enhancement ofADCC activity have been modified to comprise at least one amino acidmodification, so that their binding to FcγR has been diminished, mostpreferably abolished. In some embodiments, the antibodies of theinvention have been modified to comprise at least one amino acidmodification which reduces the binding of the constant domain to anactivating FcγR, e.g., FcγRIIIA, FcγRIIA, as compared to a wild typeantibody of the invention while retaining maximal FcγRIIB blockingactivity. Antibodies of the invention may be modified in accordance withany method known to one skilled in the art or disclosed herein. Anyamino acid modification which is known to disrupt effector function maybe used in accordance with the methods of the invention such as thosedisclosed in U.S. Application Ser. Nos. 60/439,498 (filed Jan. 9, 2003);and 60/456,041 (filed Mar. 19, 2003); both of which are incorporatedherein by reference in their entireties. In some embodiments, antibodiesof the invention are modified so that position 265 is modified, e.g.,position 265 is substituted with alanine. In preferred embodiments, themurine constant region of an antibody of the invention is swapped withthe corresponding human constant region comprising a substitution of theamino acid at position 265 with alanine, so that the effector functionis abolished while FcγRIIB blocking activity is maintained. A singleamino acid change at position 265 of IgG1 heavy chain has been shown tosignificantly reduce binding to FcγR based on ELISA assays and hasresulted in tumor mass reduction. (Shields et al. (2001) “HighResolution Mapping Of The Binding Site On Human IgG1 For Fc gamma RI, Fcgamma RII, Fc gamma RIII, And FcRn And Design Of IgG1 Variants WithImproved Binding To The Fc gamma R,” J. Biol. Chem. 276(9):6591-604; andClynes et al. (2000) “Inhibitory Fc Receptors Modulate In VivoCytoxicity Against Tumor Targets,” Nature Medicine 6(4):443-446; whichis incorporated herein by reference in its entirety.) In otherembodiments, antibodies of the invention are modified so that position297 is modified, e.g., position 297 is substituted with glutamine, sothat the N-linked glycosylation site is eliminated (see, e.g., Jefferiset al. (1995) “Recognition Sites On Human Igg For Fc Gamma Receptors:The Role Of Glycosylation,” Immunol. Lett. 44(2-3):111-117; Lund et al.(1996) “Multiple Interactions Of Igg With Its Core Oligosaccharide CanModulate Recognition By Complement And Human Fc Gamma Receptor I AndInfluence The Synthesis Of Its Oligosaccharide Chains,” J. Immunol.,157:4963-4969; Wright et al. (1994) “Effect Of Altered CH2-AssociatedCarbohydrate Structure On The Functional Properties And In Vivo Fate OfChimeric Mouse-Human Immunoglobulin G1,” J. Exp. Med. 180:1087-1096;White et al. (1997) “Ig N-glycan Orientation Can Influence InteractionsWith The Complement System,” J. Immunol. 158:426-435; all of which areincorporated herein by reference in their entireties. Modification atthis site has been reported to abolish all interaction with FcγRs. Inpreferred embodiments, the murine constant region of an antibody of theinvention is swapped with the corresponding human constant regioncomprising a substitution of the amino acid at position 265 and/or 297,so that the effector function is abolished while FcγRIIB blockingactivity is maintained.

An exemplary assay for determining the ADCC activity of the tumorspecific antibodies in the presence and absence of the antibodies of theinvention is a non-radioactive europium based fluorescent assay (BATDA,Perkin Elmer) and may comprise the following: labeling the targets cellswith an acteoxylmethyl ester of fluorescence-enhancing ester that formsa hydrophilic ligand (TDA) with the membrane of cells by hydrolysis ofthe esters; this complex is unable to leave the cell and is releasedonly upon lysis of the cell by the effectors; adding the labeled targetsto the effector cells in presence of anti-tumor antibodies and anantibody of the invention; incubating the mixture of the target andeffector cells a for 6 to 16 hours, preferably at 37° C. The extent ofADCC activity can be assayed by measuring the amount of ligand that isreleased and interacts with europium (DELFIA reagent; PerkinElmer). Theligand and the europium form a very stable and highly fluorescentchelate (EuTDA) and the measured fluorescence is directly proportionalto the number of cells lysed. Percent specific lysis can be calculatedusing the formula: (Experimental lysis-antibody-independentlysis/maximal lysis antibody-independent lysis×100%).

In some embodiments, if the sensitivity of the fluorescence-based ADCCassay is too low to detect ADCC activity of the therapeutic antibodies,the invention encompasses radioactive-based ADCC assays, such as ⁵¹Crrelease assay. Radioactive-based assays may be done instead of or incombination with fluorescent-based ADCC assays.

An exemplary ⁵¹Cr release assay for characterizing the antibodies of theinvention can comprise the following: labeling 1-2×10⁶ target cells suchas OVCAR-3 cells with ⁵¹Cr; opsonizing the target cells with antibodies4D5 and CC49 in the presence and absence of an antibody of the inventionand adding 5×10³ cells to 96 well plate. Preferably 4D5 and CC49 are ata concentration varying from 1-15 μg/mL; adding the opsonized targetcells to monocyte-derived macrophages (MDM) (effector cells); preferablyat a ratio varying from 10:1 to 100:1; incubating the mixture of cellsfor 16-18 hours at 37° C.; collecting supernatants; and analyzing theradioactivity in the supernatant. The cytotoxicity of 4D5 and CC49 inthe presence and absence of an antibody of the invention can then bedetermined, for example using the following formula percent specificlysis=(experimental lysis−antibody independent lysis/maximallysis−antibody independent lysis)×100%.

In some embodiments, the in vivo activity of the FcγRIIB antibodies ofthe invention is determined in xenograft human tumor models. Tumors maybe established using any of the cancer cell lines described supra. Insome embodiments, the tumors will be established with two cancer celllines, wherein the first cancer cell line is characterized by a lowexpression of a cancer antigen and a second cancer cell line, whereinthe second cancer cell line is characterized by a high expression of thesame cancer antigen. Tumor clearance may then be determined usingmethods known to one skilled in the art, using an anti-tumor antibodywhich immunospecifically binds the cancer antigen on the first andsecond cancer cell line, and an appropriate mouse model, e.g., a Balb/cnude mouse model (e.g., Jackson Laboratories, Taconic), with adoptivelytransferred human monocytes and MDMs as effector cells. Any of theantibodies described supra may then be tested in this animal model toevaluate the role of anti-FcγRIIB antibody of the invention in tumorclearance. Mice that may be used in the invention include for exampleFcγRIII−/− (where FcγRIIIA is knocked out); Fcγ−/− nude mice (whereFcγRI and FcγRIIIA are knocked out); or human FcγRIIB knock in mice or atransgenic knock-in mice, where mouse fcgr2 and fcgr3 loci on chromosome1 are inactivated and the mice express human FcγRIIA, human FcγRIIAhuman FcγRIIB, human FcγRIIC, human FcγRIIIA, and human FcγRIIB.

An exemplary method for testing the in vivo activity of an antibody ofthe invention may comprise the following: establishing a xenograftmurine model using a cancer cell line characterized by the expression ofa cancer antigen and determining the effect of an antibody of theinvention on an antibody specific for the cancer antigen expressed inthe cancer cell line in mediating tumor clearance. Preferably, the invivo activity is tested parallel using two cancer cell lines, whereinthe first cancer cell line is characterized by a first cancer antigenexpressed at low levels and a second cancer cell line, characterized bythe same cancer antigen expressed at a higher level relative to thefirst cancer cell line. These experiments will thus increase thestringency of the evaluation of the role of an antibody of the inventionin tumor clearance. For example, tumors may be established with theIGROV-1 cell line and the effect of an anti-FcγRIIB antibody of theinvention in tumor clearance of a Her2/neu specific antibody may beassessed. In order to establish the xenograft tumor models, 5×10⁶ viablecells, e.g., IGROV-1, SKBR3, may be injected, e.g., s.c. into mice,e.g., 8 age and weight matched female nude athymic mice using forexample Matrigel (Becton Dickinson). The estimated weight of the tumormay be determined by the formula: length×(width)²/2; and preferably doesnot exceed 3 grams. Injection of IGROV-1 cells s.c. gives rise to fastgrowing tumors while the i.p. route induces a peritoneal carcinomatosiswhich kills mice in 2 months (Benard et al. (1985) “Characterization OfA Human Ovarian Adenocarcinoma Line, IGROV1, In Tissue Culture And InNude Mice,” Cancer Research, 45:4970-4979). Since the IGROV-1 cells formtumors within 5 weeks, at day 1 after tumor cell injection, monocytes aseffectors are co-injected i.p. along with a therapeutic antibodyspecific for Her2/neu, e.g., Ch4D5, and an antibody of the invention;e.g. chimeric 2B6 or 3H7 as described supra. Preferably, the antibodiesare injected at 4 μg each per gram of mouse body weight (mbw). Theinitial injection will be followed by weekly injections of antibodiesfor 4-6 weeks thereafter at 2 μg/wk. Human effector cells will bereplenished once in 2 weeks. A group of mice will receive no therapeuticantibody but will be injected with a chimeric 4D5 comprising a N297Amutation and human IgG1 as isotype control antibodies for the anti-tumorand anti-FcγRIIB antibodies, respectively. Mice may be placed in groupsof 4 and monitored three times weekly.

Table 3 below is an exemplary setup for tumor clearance studies inaccordance with the invention. As shown in Table 3, six groups of 8 miceeach will be needed for testing the role of an antibody of the inventionin tumor clearance, wherein one target and effector cell combination isused and wherein two different combinations of the antibodyconcentration are used. In group A, only tumor cells are injected; ingroup B tumor cells and monocytes are injected; in group C, tumor cells,monocytes, an anti-tumor antibody (ch4D5) are injected; in group D,tumor cells, monocytes, anti-tumor antibody, and an anti-FcγRII antibodyare injected; in group E, tumor cells, monocytes and an anti-FcγRIIBantibody are injected; in group F, tumor cells, monocytes, Ch4D5(N297Q), and human IgG1 are injected. It will be appreciated by oneskilled in the art that various antibody concentrations of variousantibody combinations may be tested in the tumor models described.Preferably, studies using a breast cancer cell line, e.g., SKBR3, iscarried out in parallel to the above-described experiment.

TABLE 3 Exemplary Experimental Set Up In Mice ch4D5 ch2B6 Human ch4D5(N297Q at (N297Q at (IgG1 4 μg/gm 8 mice Tumor (4 μg/gm 4 μg/gm of 4μg/gm of per cell s.c Monocytes of mbw mbw day 1 of mbw mbw day 1 groupday 0 i.p at day 1 day 1 i.p.) i.p.) day 1 i.p.) i.p.) A + − − − − −B + + − − − − C + + + − − − D + + + − + − E + + − − + − F + + − + − +

The endpoint of the xenograft tumor models is determined based on thesize of the tumors, weight of mice, survival time and histochemical andhistopathological examination of the cancer, using methods known to oneskilled in the art. Each of the groups of mice in Table 3 will beevaluated. Mice are preferably monitored three times a week. Criteriafor tumor growth may be abdominal distention, presence of palpable massin the peritoneal cavity. Preferably, estimates of tumor weight versusdays after inoculation will be calculated. A comparison of theaforementioned criteria of mice in Group D compared to those in othergroups will define the role of an antibody of the invention inenhancement of tumor clearance. Preferably, antibody-treated animalswill be under observation for an additional 2 months after the controlgroup.

In alternative embodiments, human FcγRIIB “knock in” mice expressinghuman FcγRIIB on murine effector cells may be used in establishing thein vivo activity of the antibodies of the invention, rather thanadoptively transferring effector cells. Founder mice expressing thehuman FcγRIIB may be generated by “knocking in” the human FcγRIIB ontothe mouse FcγRIIB locus. The founders can then be back-crossed onto thenude background and will express the human FcγRIIB receptor. Theresulting murine effector cells will express endogenous activating FcγRIand FcγRIIIA and inhibitory human FcγRIIB receptors.

The in vivo activity of the antibodies of the invention may be furthertested in a xenograft murine model with human primary tumor derivedcells, such as human primary ovarian and breast carcinoma derived cells.Ascites and pleural effusion samples from cancer patients may be testedfor expression of Her2/neu, using methods known to one skilled in theart. Samples from ovarian carcinoma patients may be processed byspinning down the ascites at 6370 g for 20 minutes at 4° C., lysing thered blood cells, and washing the cells with PBS. Once the expression ofHer2/neu in tumor cells is determined, two samples, a median and a highexpressor may be selected for s.c. inoculation to establish thexenograft tumor model. The isolated tumor cells will then be injectedi.p. into mice to expand the cells. Approximately 10 mice may beinjected i.p. and each mouse ascites further passaged into two mice toobtain ascites from a total of 20 mice which can be used to inject agroup of 80 mice. Pleural effusion samples may be processed using asimilar method as ascites. The Her2/neu+ tumor cells from pleuraleffusion samples may be injected into the upper right & left mammarypads of the mice.

In some embodiments, if the percentage of neoplastic cells in theascites or pleural effusion samples is low compared to other cellularsubsets, the neoplastic cells may be expanded in vitro. In otherembodiments, tumor cells may be purified using CC49 antibody(anti-TAG-72)-coated magnetic beads as described previously, see, e.g.,Barker et al. (2001) “An Immunomagnetic-Based Method For ThePurification Of Ovarian Cancer Cells From Patient-Derived Ascites,”Gynecol. Oncol. 82:57-63, which is incorporated herein by reference inits entirety. Briefly, magnetic beads coated with CC49 antibody can beused to separate the ovarian tumor cells that will be detached from thebeads by an overnight incubation at 37° C. In some embodiments, if thetumor cells lack the TAG-72 antigen, negative depletion using a cocktailof antibodies, such as those provided by Stem Cell Technologies, Inc.,Canada, may be used to enrich the tumor cells.

In other embodiments, other tumors markers besides Her2/neu may be usedto separate tumor cells obtained from the ascites and pleural effusionsamples from non-tumor cells. In the case of pleural effusion or breasttissue, it has been recently reported that CD44 (an adhesion molecule),B38.1 (a breast/ovarian cancer-specific marker), CD24 (an adhesionmolecule) may be used as markers, see, e.g., Al Hajj, et al. (2003)“Prospective Identification Of Tumorigenic Breast Cancer Cells,” Proc.Natl. Acad. Sci. USA 100:3983-3988; which is incorporated herein byreference in its entirety. Once tumor cells are purified they may beinjected s.c. into mice for expansion.

Preferably, immunohistochemistry and histochemistry is performed onascites and pleural effusion of patients to analyze structuralcharacteristics of the neoplasia. Such methods are known to one skilledin the art and encompassed within the invention. The markers that may bemonitored include for example cytokeratin (to identify ovarianneoplastic and mesothelial cells from inflammatory and mesenchymalcells); calretinin (to separate mesothelial from Her2neu positiveneoplastic cells); and CD45 (to separate inflammatory cells from therest of the cell population in the samples). Additional markers that maybe followed include CD3 (T cells), CD20 (B cells), CD56 (NK cells), andCD14 (monocytes). It will be appreciated by one skilled in the art thatthe immunohistochemistry and histochemistry methods described supra, areanalogously applied to any tumor cell for use in the methods of theinvention. After s.c. inoculation of tumor cells, mice are followed forclinical and anatomical changes. As needed, mice may be necropsied tocorrelate total tumor burden with specific organ localization.

In a specific embodiment, tumors are established using carcinoma celllines such as IGROV-1, OVCAR-8, SK-B, and OVCAR-3 cells and humanovarian carcinoma ascites and pleural effusion from breast cancerpatients. The ascites preferably contain both the effectors and thetumor targets for the antibodies being tested. Human monocytes will betransferred as effectors.

The in vivo activity of the antibodies of the invention may also betested in an animal model, e.g., Balb/c nude mice, injected with cellsexpressing FcγRIIB, including but not limited to SK-BR-3 with ATCCaccession number HTB-30 (see, e.g., Tremp et al. (1976) “Human BreastCancer In Culture,” Recent Results Cancer Res. 57:33-41); B-lymphocytes;cells derived from Burkitts lymphoma, e.g., Raji cells with ATCCaccession number CCL-86 (see, e.g., Epstein et al. (1965)“Characteristics And Mode Of Growth Of Tissue Culture Strain (EB1) OfHuman Lymphoblasts From Burkitt's Lymphoma,” J. Natl. Cancer Inst. 34:231-240), Daudi cells with ATCC accession number CCL-213 (see, e.g.,Klein et al. (1968) “Surface IgM-Kappa Specificity On A Burkitt LymphomaCell In Vivo And In Derived Culture Lines,” Cancer Res. 28: 1300-1310);ovarian carcinoma cell lines, e.g., OVCAR-3 with ATCC accession numberHTB-161 (see, e.g., Hamilton et al. (1983) “Characterization Of A HumanOvarian Carcinoma Cell Line (NIH:OVCAR-3) With Androgen And EstrogenReceptors,” Cancer Res. 43(11):5379-5389), SK-OV-3, PA-1, CAOV3, OV-90,and IGROV-1 (available from the NCl repository; Benard et al. (1985)“Characterization Of A Human Ovarian Adenocarcinoma Line, IGROV1, InTissue Culture And In Nude Mice,” Cancer Research, 45:4970-4979; whichis incorporated herein by reference in its entirety.

An exemplary assay for measuring the in vivo activity of the antibodiesof the invention may comprise the following: Balb/c Nude female mice(Taconic, MD) are injected at day 0 with cells expressing FcγRIIB suchas 5×10⁶ Daudi cells for example by the subcutaneous route. Mice (e.g.,5 mice per group) also receive i.p. injection of PBS (negative control),ch 4.4.20 (anti-FITC antibody) as a negative control, and as a positivecontrol another therapeutic cancer antibody such as those disclosedherein, e.g., RITUXAN® (rituximab), (e.g., at 10 μg/g) or 10 μg/g ch2B6once a week starting at day 0. Mice are observed, e.g., twice a weekfollowing injection, and tumor size (length and width) is determinedusing for example a caliper. Tumor weight in mg is estimated using theformula: (length×width²)/2.

Preferably, the antibodies of the invention have an enhanced efficacy indecreasing tumor relative to a cancer therapeutic antibody whenadministered at the same dose, e.g., 10 μg/g, over a time period of atleast 14 days, at least 21 days, at least 28 days, or at least 35 days.In most preferred embodiments, the antibodies of the invention reducetumor size by at least 10 fold, at least 100 fold, at least 1000 foldrelative to administration of a cancer therapeutic antibody at the samedose. In yet another preferred embodiment, the antibodies of theinvention completely abolish the tumor.

IV. Polynucleotides Encoding an Antibody

The present invention also includes polynucleotides that encode theantibodies of the invention (e.g., mouse monoclonal antibody producedfrom clone 2B6 or 3H7, with ATCC accession numbers PTA-4591 andPTA-4592, respectively), or other monoclonal antibodies produced byimmunization methods of the invention, and humanized versions thereof,and methods for producing same.

The present invention encompass the polynucleotide encoding the heavychain of the 2B6 antibody, with ATCC accession number PTA-4591, asdisclosed in SEQ ID NO:27. The present invention also encompasses thepolynucleotide encoding the light chain of the 2B6 antibody with ATCCaccession number PTA-4591, as disclosed in SEQ ID NO:25.

SEQ ID NO: 27: caggtccaat tgcagcagcc tgtgactgag ctggtgaggc cgggggcttc 50 agtgatgttg tcctgcaagg cttctgacta ccccttcacc aactactgga 100tacactgggt aaagcagagg cctggacaag gcctggagtg gatcggagtg 150 attgatccttctgatactta tccaaattac aataaaaagt tcaagggcaa 200 ggccacattg actgtagtcgtatcctccag cacagcctac atgcagctca 250 gcagcctgac atctgacgat tctgcggtctattactgtgc aagaaacggt 300 gattccgatt attactctgg tatggactac tggggtcaaggaacctcagt 350 caccgtctcc tca 363 SEQ ID NO: 25: gacatcttgc tgactcagtctccagccatc ctgtctgtga gtccaggaga  50 gagagtcagt ttttcctgca ggaccagtcagagcattggc acaaacatac 100 actggtatca gcaaagaaca aatggttttc caaggcttctcataaagaat 150 gtttctgagt ctatctctgg gatcccttcc aggtttagtg gcagtggatc200 agggacagat tttattctta gcatcaacag tgtggagtct gaagatattg 250cagattatta ttgtcaacaa agtaatacct ggccgttcac gttcggaggg 300 gggaccaagctggaaataaa a 321

The methods of the invention also encompass polynucleotides thathybridize under various stringency, e.g., high stringency, intermediateor lower stringency conditions, to polynucleotides that encode anantibody of the invention. The hybridization can be performed undervarious conditions of stringency. By way of example and not limitation,procedures using conditions of low stringency are as follows (see alsoShilo et al. (1981) “DNA Sequences Homologous To Vertebrate OncogenesAre Conserved In Drosophila Melanogaster,” Proc. Natl. Acad. Sci. U.S.A78, 6789 6792). Filters containing DNA are pretreated for 6 h at 40° C.in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5),5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmonsperm DNA. Hybridizations are carried out in the same solution with thefollowing modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/mlsalmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20×10⁶ cpm³²P-labeled probe is used. Filters are incubated in hybridizationmixture for 18-20 h at 40° C., and then washed for 1.5 h at 55° C. in asolution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1%SDS. The wash solution is replaced with fresh solution and incubated anadditional 1.5 h at 60° C. Filters are blotted dry and exposed forautoradiography. If necessary, filters are washed for a third time at65-68° C. and re-exposed to film. Other conditions of low stringencywhich may be used are well known in the art (e.g., as employed forcross-species hybridizations). By way of example and not limitation,procedures using conditions of high stringency are as follows.Prehybridization of filters containing DNA is carried out for 8 h toovernight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/mldenatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C. inprehybridization mixture containing 100 μg/ml denatured salmon sperm DNAand 5-20×10⁶ cpm of ³²P-labeled probe. Washing of filters is done at 37°C. for 1 h in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and0.01% BSA. This is followed by a wash in 0.1×SSC at 50° C. for 45 minbefore autoradiography. Other conditions of high stringency which may beused are well known in the art. Selection of appropriate conditions forsuch stringencies is well known in the art (see e.g., Sambrook et al.,1989, MOLECULAR CLONING, A LABORATORY MANUAL, 2d Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; see also, Ausubel et al.,eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY series of laboratorytechnique manuals, 1987-1997, Current Protocols, 1994-1997 John Wileyand Sons, Inc.; see especially, Dyson, 1991, “Immobilization of nucleicacids and hybridization analysis,” In: ESSENTIAL MOLECULAR BIOLOGY: APRACTICAL APPROACH, Vol. 2, T. A. Brown, ed., pp. 111-156, IRL Press atOxford University Press, Oxford, UK).

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art.

A polynucleotide encoding an antibody may be generated from nucleic acidfrom a suitable source (e.g., a cDNA library generated from, or nucleicacid, preferably poly A+ RNA, isolated from, any tissue or cellsexpressing the antibody, such as hybridoma cells selected to express anantibody of the invention, e.g., 2B6 or 3H7) by hybridization with Igspecific probes and/or PCR amplification using synthetic primershybridizable to the 3′ and 5′ ends of the sequence or by cloning usingan oligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence of the antibody is determined, thenucleotide sequence of the antibody may be manipulated using methodswell known in the art for the manipulation of nucleotide sequences,e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.(see, for example, the techniques described in Sambrook et al., 1989,MOLECULAR CLONING, A LABORATORY MANUAL, 2d Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; see also, Ausubel et al.,eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY series of laboratorytechnique manuals, 1987-1997, Current Protocols, 1994-1997 John Wileyand Sons, Inc., which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, one or more of the CDRs are inserted withinframework regions using routine recombinant DNA techniques. Theframework regions may be naturally occurring or consensus frameworkregions, and preferably human framework regions (see, e.g., Chothia etal. (1998) “Structural Determinants In The Sequences Of ImmunoglobulinVariable Domain,” J. Mol. Biol. 278: 457-479 for a listing of humanframework regions). Preferably, the polynucleotide generated by thecombination of the framework regions and CDRs encodes an antibody thatspecifically binds to FcγRIIB with greater affinity than said antibodybinds FcγRIIA. Preferably, as discussed supra, one or more amino acidsubstitutions may be made within the framework regions, and, preferably,the amino acid substitutions improve binding of the antibodies of theinvention to FcγRIIB. Representative plasmids, pMG×608 (pCI-neo[Invitrogen, Inc.] containing a humanized 2B6 heavy chain with humanVH1-18 and JH6 germline sequences as frameworks, 2B6 mouse CDRs andhuman IgG, Fc constant region) and pMG×611 (pCI-neo containing ahumanized 2B6 light chain with human VK-A26 and JK4 as frameworks, humankappa as constant region, and mouse 2B6 light chain CDRs with N₅₀→Y andV₅₁→A in CDR2), having ATCC Accession numbers PTA-5963 and PTA-5964,respectively, were deposited under the provisions of the Budapest Treatywith the American Type Culture Collection (10801 University Blvd.,Manassas, Va. 20110-2209) on May 7, 2004, respectively, and areincorporated herein by reference.

In another embodiment, human libraries or any other libraries availablein the art, can be screened by standard techniques known in the art, toclone the nucleic acids encoding the antibodies of the invention.

V. Recombinant Expression of Antibodies

Once a nucleic acid sequence encoding an antibody of the invention hasbeen obtained, the vector for the production of the antibody may beproduced by recombinant DNA technology using techniques well known inthe art. Methods which are well known to those skilled in the art can beused to construct expression vectors containing the antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.(See, for example, the techniques described in Sambrook et al., 1990,MOLECULAR CLONING, A LABORATORY MANUAL, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. and Ausubel et al. eds., 1998,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY).

An expression vector comprising the nucleotide sequence of an antibodycan be transferred to a host cell by conventional techniques (e.g.,electroporation, liposomal transfection, and calcium phosphateprecipitation) and the transfected cells are then cultured byconventional techniques to produce the antibody of the invention. Inspecific embodiments, the expression of the antibody is regulated by aconstitutive, an inducible or a tissue, specific promoter.

The host cells used to express the recombinant antibodies of theinvention may be either bacterial cells such as Escherichia coli, or,preferably, eukaryotic cells, especially for the expression of wholerecombinant immunoglobulin molecule. In particular, mammalian cells suchas Chinese hamster ovary cells (CHO), in conjunction with a vector suchas the major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for immunoglobulins(Cockett et al. (1990) “High Level Expression Of Tissue Inhibitor OfMetalloproteinases In Chinese Hamster Ovary Cells Using GlutamineSynthetase Gene Amplification,” Biotechnology 8:662-667).

A variety of host-expression vector systems may be utilized to expressthe antibodies of the invention. Such host-expression systems representvehicles by which the coding sequences of the antibodies may be producedand subsequently purified, but also represent cells which may, whentransformed or transfected with the appropriate nucleotide codingsequences, express the antibodies of the invention in situ. Theseinclude, but are not limited to, microorganisms such as bacteria (e.g.,E. coli and B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing immunoglobulincoding sequences; yeast (e.g., Saccharomyces Pichia) transformed withrecombinant yeast expression vectors containing immunoglobulin codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the immunoglobulincoding sequences; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus (CaMV) and tobaccomosaic virus (TMV)) or transformed with recombinant plasmid expressionvectors (e.g., Ti plasmid) containing immunoglobulin coding sequences;or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells,lymphotic cells (see U.S. Pat. No. 5,807,715), Per C.6 cells (ratretinal cells developed by Crucell)) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodybeing expressed. For example, when a large quantity of such a protein isto be produced, for the generation of pharmaceutical compositions of anantibody, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include, but are not limited, to the E. coli expression vectorpUR278 (Ruther et al. (1983) “Easy Identification Of cDNA Clones,” EMBOJ. 2:1791-1794), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye et al. (1985)“Up-Promoter Mutations In The Lpp Gene Of Escherichia coli,” NucleicAcids Res. 13:3101-3110; Van Heeke et al. (1989) “Expression Of HumanAsparagine Synthetase In Escherichia coli,” J. Biol. Chem. 24:5503-5509;and the like. pGEX vectors may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption and binding to a matrixglutathione-agarose beads followed by elution in the presence of freegluta-thione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (e.g., the polyhedrin gene) ofthe virus and placed under control of an AcNPV promoter (e.g., thepolyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the immunoglobulin molecule in infected hosts. (e.g., seeLogan et al. (1984) “Adenovirus Tripartite Leader Sequence EnhancesTranslation Of mRNAs Late After Infection,” Proc. Natl. Acad. Sci. USA81:3655-3659). Specific initiation signals may also be required forefficient translation of inserted antibody coding sequences. Thesesignals include the ATG initiation codon and adjacent sequences.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (seeBitter et al. (1987) “Expression And Secretion Vectors For Yeast,”Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 andHs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably express anantibody of the invention may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express theantibodies of the invention. Such engineered cell lines may beparticularly useful in screening and evaluation of compounds thatinteract directly or indirectly with the antibodies of the invention.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al. (1977)“Transfer Of Purified Herpes Virus Thymidine Kinase Gene To CulturedMouse Cells,” Cell 11:223-232), hypoxanthine-guaninephosphoribosyltransferase (Szybalska et al. (1962) “Genetics Of HumanCess Line. IV. DNA-Mediated Heritable Transformation Of A BiochemicalTrait,” Proc. Natl. Acad. Sci. USA 48:2026-2034), and adeninephosphoribosyltransferase (Lowy et al. (1980) “Isolation Of TransformingDNA: Cloning The Hamster aprt Gene,” Cell 22:817-823) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al. (1980) “Transformation Of Mammalian Cells With An AmplifiableDominant-Acting Gene,” Proc. Natl. Acad. Sci. USA 77:3567-3570; O'Hareet al. (1981) “Transformation Of Mouse Fibroblasts To MethotrexateResistance By A Recombinant Plasmid Expressing A ProkaryoticDihydrofolate Reductase,” Proc. Natl. Acad. Sci. USA 78:1527-1531); gpt,which confers resistance to mycophenolic acid (Mulligan et al. (1981)“Selection For Animal Cells That Express The Escherichia coli GeneCoding For Xanthine-Guanine Phosphoribosyltransferase,” Proc. Natl.Acad. Sci. USA 78:2072-2076); neo, which confers resistance to theaminoglycoside G-418 (Tachibana et al. (1991) “Altered Reactivity OfImmunoglobulin Produced By Human-Human Hybridoma Cells Transfected BypSV2-Neo Gene,” Cytotechnology 6(3):219-226; Tolstoshev (1993) “GeneTherapy, Concepts, Current Trials And Future Directions,” Ann. Rev.Pharmacol. Toxicol. 32:573-596; Mulligan (1993) “The Basic Science OfGene Therapy,” Science 260:926-932; and Morgan et al. (1993) “Human genetherapy,” Ann. Rev. Biochem. 62:191-217). Methods commonly known in theart of recombinant DNA technology which can be used are described inAusubel et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley & Sons, NY; Kriegler, 1990, GENE TRANSFER AND EXPRESSION, ALABORATORY MANUAL, Stockton Press, NY; and in Chapters 12 and 13,Dracopoli et al. (eds), 1994, CURRENT PROTOCOLS IN HUMAN GENETICS, JohnWiley & Sons, NY.; Colbere-Garapin et al. (1981) “A New Dominant HybridSelective Marker For Higher Eukaryotic Cells,” J. Mol. Biol. 150:1-14;and hygro, which confers resistance to hygromycin (Santerre et al.(1984) “Expression Of Prokaryotic Genes For Hygromycin B And G418Resistance As Dominant-Selection Markers In Mouse L Cells,” Gene30:147-156).

The expression levels of an antibody of the invention can be increasedby vector amplification (for a review, see Bebbington and Hentschel,“The use of vectors based on gene amplification for the expression ofcloned genes in mammalian cells,” in DNA CLONING, Vol. 3. (AcademicPress, New York, 1987)). When a marker in the vector system expressingan antibody is amplifiable, increase in the level of inhibitor presentin culture of host cell will increase the number of copies of the markergene. Since the amplified region is associated with the nucleotidesequence of the antibody, production of the antibody will also increase(Crouse et al. (1983) “Expression And Amplification Of Engineered MouseDihydrofolate Reductase Minigenes,” Mol. Cell. Biol. 3:257-266).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot (1986) “Expression And Amplification Of Engineered MouseDihydrofolate Reductase Minigenes,” Nature 322:562-565; Kohler (1980)“Immunoglobulin Chain Loss In Hybridoma Lines,” Proc. Natl. Acad. Sci.USA 77:2197-2199). The coding sequences for the heavy and light chainsmay comprise cDNA or genomic DNA.

Once the antibody of the invention has been recombinantly expressed, itmay be purified by any method known in the art for purification of anantibody, for example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antigen after Protein A, andsizing column chromatography), centrifugation, differential solubility,or by any other standard technique for the purification of proteins.

VI. Prophylactic and Therapeutic Methods

The present invention encompasses antibody-based therapies which involveadministering one or more of the antibodies of the invention to ananimal, preferably a mammal, and most preferably a human, forpreventing, treating, or ameliorating symptoms associated with adisease, disorder, or infection, associated with aberrant levels oractivity of FcγRIIB and/or treatable by altering immune functionassociated with FcγRIIB activity or enhancing cytotoxic activity of asecond therapeutic antibody or enhancing efficacy of a vaccinecomposition or breaking tolerance to an antigen. In some embodiments,therapy by administration of one or more antibodies of the invention iscombine with administration of one or more therapies such as, but notlimited to, chemotherapies, radiation therapies, hormonal therapies,and/or biological therapies/immunotherapies

Prophylactic and therapeutic compounds of the invention include, but arenot limited to, proteinaceous molecules, including, but not limited to,peptides, polypeptides, proteins, including post-translationallymodified proteins, antibodies, etc.; small molecules (less than 1000daltons), inorganic or organic compounds; nucleic acid moleculesincluding, but not limited to, double-stranded or single-stranded DNA,double-stranded or single-stranded RNA, as well as triple helix nucleicacid molecules. Prophylactic and therapeutic compounds can be derivedfrom any known organism (including, but not limited to, animals, plants,bacteria, fungi, and protista, or viruses) or from a library ofsynthetic molecules.

Antibodies may be provided in pharmaceutically acceptable compositionsas known in the art or as described herein. As detailed below, theantibodies of the invention can be used in methods of treating cancer(particularly to enhance passive immunotherapy or efficacy of a cancervaccine) or allergies (e.g., to enhance efficacy of a vaccine fortreatment of allergy).

Antibodies of the present invention that function as a prophylactic andor therapeutic agent of a disease, disorder, or infection can beadministered to an animal, preferably a mammal and most preferably ahuman, to treat, prevent or ameliorate one or more symptoms associatedwith the disease, disorder, or infection. Antibodies of the inventioncan be administered in combination with one or more other prophylacticand/or therapeutic agents useful in the treatment, prevention ormanagement of a disease, disorder, or infection associated with aberrantlevels or activity of FcγRIIB and/or treatable by altering immunefunction associated with FcγRIIB activity. In certain embodiments, oneor more antibodies of the invention are administered to a mammal,preferably a human, concurrently with one or more other therapeuticagents useful for the treatment of cancer. The term “concurrently” isnot limited to the administration of prophylactic or therapeutic agentsat exactly the same time, but rather it is meant that antibodies of theinvention and the other agent are administered to a subject in asequence and within a time interval such that the antibodies of theinvention can act together with the other agent to provide an increasedbenefit than if they were administered otherwise. For example, eachprophylactic or therapeutic agent may be administered at the same timeor sequentially in any order at different points in time; however, ifnot administered at the same time, they should be administeredsufficiently close in time so as to provide the desired therapeutic orprophylactic effect. Each therapeutic agent can be administeredseparately, in any appropriate form and by any suitable route.

In various embodiments, the prophylactic or therapeutic agents areadministered less than 1 hour apart, at about 1 hour apart, at about 1hour to about 2 hours apart, at about 2 hours to about 3 hours apart, atabout 3 hours to about 4 hours apart, at about 4 hours to about 5 hoursapart, at about 5 hours to about 6 hours apart, at about 6 hours toabout 7 hours apart, at about 7 hours to about 8 hours apart, at about 8hours to about 9 hours apart, at about 9 hours to about 10 hours apart,at about 10 hours to about 11 hours apart, at about 11 hours to about 12hours apart, no more than 24 hours apart or no more than 48 hours apart.In preferred embodiments, two or more components are administered withinthe same patient visit.

The dosage amounts and frequencies of administration provided herein areencompassed by the terms therapeutically effective and prophylacticallyeffective. The dosage and frequency further will typically varyaccording to factors specific for each patient depending on the specifictherapeutic or prophylactic agents administered, the severity and typeof cancer, the route of administration, as well as age, body weight,response, and the past medical history of the patient. Suitable regimenscan be selected by one skilled in the art by considering such factorsand by following, for example, dosages reported in the literature andrecommended in the Physician's Desk Reference (56^(th) ed., 2002).

The antibodies of this invention may also be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), which, for example, serve to increase the number or activityof effector cells which interact with the antibodies and, increaseimmune response. The antibodies of this invention may also beadvantageously utilized in combination with one or more drugs used totreat a disease, disorder, or infection such as, for example anti-canceragents or anti-viral agents, e.g., as detailed below.

A. Cancers

Antibodies of the invention can be used alone or in combination withother therapeutic antibodies known in the art to prevent, inhibit orreduce the growth of primary tumores or metastasis of cancerous cells.In one embodiment, antibodies of the invention can be used incombination with antibodies used in cancer immunotherapy. The inventionencompasses the use of the antibodies of the invention in combinationwith another therapeutic antibody to enhance the efficacy of suchimmunotherapy by increasing the potency of the therapeutic antibody'seffector function, e.g., ADCC, CDC, phagocytosis, opsonization, etc.Although not intending to be bound by a particular mechanism of actionantibodies of the invention block FcγRIIB, preferably on monocytes andmacrophages and thus enhance the therapeutic benefits a clinicalefficacy of tumor specific antibodies by, for example, enhancingclearance of the tumors mediated by activating fcγRs. Accordingly, theinvention provides methods of preventing or treating cancercharacterized by a cancer antigen, when administered in combination withanother antibody that specifically binds a cancer antigen and iscytotoxic. The antibodies of the invention are useful for prevention ortreatment of cancer, particularly in potentiating the cytotoxic activityof cancer antigen-specific therapeutic antibodies with cytotoxicactivity to enhance tumor cell killing by the antibodies of theinvention and/or enhancing for example, ADCC activity or CDC activity ofthe therapeutic antibodies. In a specific embodiment, an antibody of theinvention, when administered alone or in combination with a cytotoxictherapeutic antibody, inhibits or reduces the growth of primary tumor ormetastasis of cancerous cells by at least 99%, at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, at least 50%, at least 45%, at least 40%, at least 45%, at least35%, at least 30%, at least 25%, at least 20%, or at least 10% relativeto the growth of primary tumor or metastasis in absence of said antibodyof the invention. In a preferred embodiment, antibodies of the inventionin combination with a cytotoxic therapeutic antibody inhibit or reducethe growth of primary tumor or metastasis of cancer by at least 99%, atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 60%, at least 50%, at least 45%, at least 40%, atleast 45%, at least 35%, at least 30%, at least 25%, at least 20%, or atleast 10% relative to the growth or metastasis in absence of saidantibodies.

The transition from a normal to a malignant state is a multistep processinvolving genetic and epigenetic changes. In fact, numerous alterationsoccur in the cellular regulatory circuits that facilitate thisprogression which enables tumor cells to evade the commitment toterminal differentiation and quiescence that normally regulate tissuehomeostasis. Certain genes have been implicated in invasiveness andmetastatic potential of cancer cells such as CSF-1 (colony stimulatingfactor 1 or macrophage colony stimulating factor). Although notintending to be bound by a particular mechanism of action, CSF-1 maymediate tumor progression and metastasis by recruiting macrophages tothe tumor site where they promote progression of tumor. It is believedthat macrophages have a trophic role in mediating tumor progression andmetastasis perhaps by the secretion of angiogenic factors, e.g.,thymidine phosphorylase, vascular endothelial-derived growth factor;secretion of growth factors such as epidermal growth factor that couldact as a paracrine factor on tumor cells, and thus promoting tumor cellmigration and invasion into blood vessels. (See, e.g., Lin et al. (2001)“Colony-Stimulating Factor 1 Promotes Progression Of Mammary Tumors ToMalignancy,” J. Exp. Med. 193(6): 727-739; Lin et al. (2002) “TheMacrophage Growth Factor Csf-1 In Mammary Gland Development And TumorProgression,” Journal of Mammary Gland Biology and Neoplasm 7(2):147-162; Scholl et al. (1993) “Is Colony-Stimulating Factor-1 A KeyMediator Of Breast Cancer Invasion And Metastasis?” MolecularCarcinogenesis, 7: 207-211; Clynes et al. (2000) “Inhibitory FcReceptors Modulate In Vivo Cytoxicity Against Tumor Targets,” NatureMedicine, 6(4): 443-446; Fidler et al. (1985) “Macrophages AndMetastasis—A Biological Approach To Cancer Therapy,” Cancer Research,45: 4714-4726).

The invention encompasses using the antibodies of the invention to blockmacrophage mediated tumor cell progression and metastasis. Theantibodies of the invention are particularly useful in the treatment ofsolid tumors, where macrophage infiltration occurs. The antagonisticantibodies of the invention are particularly useful for controlling,e.g., reducing or eliminating, tumor cell metastasis, by reducing oreliminating the population of macrophages that are localized at thetumor site. In some embodiments, the antibodies of the invention areused alone to control tumor cell metastasis. Although not intending tobe bound by a particular mechanism of action the antagonistic antibodiesof the invention, when administered alone bind the inhibitory FcγRIIB onmacrophages and effectively reduce the population of macrophages andthus restrict tumor cell progression. The antagonistic antibodies of theinvention reduce, or preferably eliminate macrophages that are localizedat the tumor site, since FcγRIIB is preferentially expressed onactivated monocytes and macrophages including tumor-infiltratingmacrophages. In some embodiments, the antibodies of the invention areused in the treatment of cancers that are characterized by theoverexpression of CSF-1, including but not limited to breast, uterine,and ovarian cancers.

The invention further encompasses antibodies that effectively deplete oreliminate immune cells other than macrophages that express FcγRIIB,e.g., dendritic cells and B-cells. Effective depletion or elimination ofimmune cells using the antibodies of the invention may range from areduction in population of the immune cells by 50%, 60%, 70%, 80%,preferably 90%, and most preferably 99%. Thus, the antibodies of theinvention have enhanced therapeutic efficacy either alone or incombination with a second antibody, e.g., a therapeutic antibody such asanti-tumor antibodies, anti-viral antibodies, and anti-microbialantibodies. In some embodiments, the therapeutic antibodies havespecificity for a cancer cell or an inflammatory cell. In otherembodiments, the second antibody binds a normal cell. Although notintending to be bound by a particular mechanism of action, when theantibodies of the invention are used alone to deplete FcγRIIB-expressingimmune cells, the population of cells is redistributed so thateffectively the cells that are remaining have the activating Fcreceptors and thus the suppression by FcγRIIB is alleviated. When usedin combination with a second antibody, e.g., a therapeutic antibody theefficacy of the second antibody is enhanced by increasing theFc-mediated effector function of the antibody.

The antibodies and fragments thereof of the invention and methods oftreatment are believed to be effective for the treatment of both liquidand solid cancers. By liquid cancers it is meant cancers of the bonemarrow, such as leukemias. Solid cancers generally refer to cancers oforgans and/or tissues. Cancers and related disorders that can be treatedor prevented by methods and compositions of the present inventioninclude, but are not limited to, the following: Leukemias including, butnot limited to, acute leukemia, acute lymphocytic leukemia, acutemyelocytic leukemias such as myeloblastic, promyelocytic,myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplasticsyndrome, chronic leukemias such as but not limited to, chronicmyelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairycell leukemia; polycythemia vera; lymphomas such as but not limited toHodgkin's disease, non-Hodgkin's disease; multiple myelomas such as butnot limited to smoldering multiple myeloma, nonsecretory myeloma,osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma andextramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonalgammopathy of undetermined significance; benign monoclonal gammopathy;heavy chain disease; bone and connective tissue sarcomas such as but notlimited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma,malignant giant cell tumor, fibrosarcoma of bone, chordoma, periostealsarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma;brain tumors including but not limited to, glioma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, nonglial tumor, acousticneurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma; breast cancer including, but notlimited to, adenocarcinoma, lobular (small cell) carcinoma, intraductalcarcinoma, medullary breast cancer, mucinous breast cancer, tubularbreast cancer, papillary breast cancer, Paget's disease, andinflammatory breast cancer; adrenal cancer, including but not limitedto, pheochromocytom and adrenocortical carcinoma; thyroid cancer such asbut not limited to papillary or follicular thyroid cancer, medullarythyroid cancer and anaplastic thyroid cancer; pancreatic cancer,including but not limited to, insulinoma, gastrinoma, glucagonoma,vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor;pituitary cancers including but not limited to, Cushing's disease,prolactin-secreting tumor, acromegaly, and diabetes insipius; eyecancers including but not limited to, ocular melanoma such as irismelanoma, choroidal melanoma, and cilliary body melanoma, andretinoblastoma; vaginal cancers, including but not limited to, squamouscell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, includingbut not limited to, squamous cell carcinoma, melanoma, adenocarcinoma,basal cell carcinoma, sarcoma, and Paget's disease; cervical cancersincluding but not limited to, squamous cell carcinoma, andadenocarcinoma; uterine cancers including but not limited to,endometrial carcinoma and uterine sarcoma; ovarian cancers including butnot limited to, ovarian epithelial carcinoma, borderline tumor, germcell tumor, and stromal tumor; esophageal cancers including but notlimited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma,mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma,plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma;stomach cancers including but not limited to, adenocarcinoma, fungating(polypoid), ulcerating, superficial spreading, diffusely spreading,malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; coloncancers; rectal cancers; liver cancers including but not limited tohepatocellular carcinoma and hepatoblastoma, gallbladder cancersincluding but not limited to, adenocarcinoma; cholangiocarcinomasincluding but not limited to, pappillary, nodular, and diffuse; lungcancers including but not limited to, non-small cell lung cancer,squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma,large-cell carcinoma and small-cell lung cancer; testicular cancersincluding but not limited to, germinal tumor, seminoma, anaplastic,classic (typical), spermatocytic, nonseminoma, embryonal carcinoma,teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancersincluding but not limited to, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma; penal cancers; oral cancers including but not limitedto, squamous cell carcinoma; basal cancers; salivary gland cancersincluding but not limited to, adenocarcinoma, mucoepidermoid carcinoma,and adenoidcystic carcinoma; pharynx cancers including but not limitedto, squamous cell cancer, and verrucous; skin cancers including but notlimited to, basal cell carcinoma, squamous cell carcinoma and melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, acral lentiginous melanoma; kidney cancers including but notlimited to, renal cell cancer, adenocarcinoma, hypernephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);Wilms' tumor; bladder cancers including but not limited to, transitionalcell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. Inaddition, cancers include myxosarcoma, osteogenic sarcoma,endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma,hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogeniccarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillarycarcinoma and papillary adenocarcinomas (for a review of such disorders,see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co.,Philadelphia and Murphy et al., 1997, Informed Decisions The CompleteBook of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin,Penguin Books U.S.A., Inc., United States of America).

Accordingly, the methods and compositions of the invention are alsouseful in the treatment or prevention of a variety of cancers or otherabnormal proliferative diseases, including (but not limited to) thefollowing: carcinoma, including that of the bladder, breast, colon,kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin;including squamous cell carcinoma; hematopoietic tumors of lymphoidlineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Berkettslymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; othertumors, including melanoma, seminoma, tetratocarcinoma, neuroblastomaand glioma; tumors of the central and peripheral nervous system,including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors ofmesenchymal origin, including fibrosafcoma, rhabdomyoscarama, andosteosarcoma; and other tumors, including melanoma, xenodermapegmentosum, keratoactanthoma, seminoma, thyroid follicular cancer andteratocarcinoma. It is also contemplated that cancers caused byaberrations in apoptosis would also be treated by the methods andcompositions of the invention. Such cancers may include but not belimited to follicular lymphomas, carcinomas with p53 mutations, hormonedependent tumors of the breast, prostate and ovary, and precancerouslesions such as familial adenomatous polyposis, and myelodysplasticsyndromes. In specific embodiments, malignancy or dysproliferativechanges (such as metaplasias and dysplasias), or hyperproliferativedisorders, are treated or prevented by the methods and compositions ofthe invention in the ovary, bladder, breast, colon, lung, skin,pancreas, or uterus. In other specific embodiments, sarcoma, melanoma,or leukemia is treated or prevented by the methods and compositions ofthe invention.

Cancers associated with the cancer antigens may be treated or preventedby administration of the antibodies of the invention in combination withan antibody that binds the cancer antigen and is cytotoxic. In oneparticular embodiment, the antibodies of the invention enhance theantibody mediated cytotoxic effect of the antibody directed at theparticular cancer antigen. For example, but not by way of limitation,cancers associated with the following cancer antigen may be treated orprevented by the methods and compositions of the invention. KS 1/4pan-carcinoma antigen (Perez et al. (1989) “Isolation AndCharacterization Of A cDNA Encoding The Ks1/4 Epithelial CarcinomaMarker,” J. Immunol. 142:3662 3667; Möller et al. (1991)“Bispecific-Monoclonal-Antibody-Directed Lysis Of Ovarian CarcinomaCells By Activated Human T Lymphocytes,” Cancer Immunol. Immunother.33(4):210-216), ovarian carcinoma antigen (CA125) (Yu et al. (1991)“Coexpression Of Different Antigenic Markers On Moieties That Bear CA125 Determinants,” Cancer Res. 51(2):468 475), prostatic acid phosphate(Tailor et al. (1990) “Nucleotide Sequence Of Human Prostatic AcidPhosphatase Determined From A Full-Length cDNA Clone,” Nucl. Acids Res.18(16):4928), prostate specific antigen (Henttu et al. (1989) “cDNACoding For The Entire Human Prostate Specific Antigen Shows HighHomologies To The Human Tissue Kallikrein Genes,” Biochem. Biophys. Res.Comm. 10(2):903 910; Israeli et al. (1993) “Molecular Cloning Of AComplementary DNA Encoding A Prostate-Specific Membrane Antigen,” CancerRes. 53:227 230), melanoma-associated antigen p97 (Estin et al. (1989)“Transfected Mouse Melanoma Lines That Express Various Levels Of HumanMelanoma-Associated Antigen p97,” J. Natl. Cancer Instit. 81(6):445454), melanoma antigen gp75 (Vijayasardahl et al. (1990) “The MelanomaAntigen Gp75 Is The Human Homologue Of The Mouse B (Brown) Locus GeneProduct,” J. Exp. Med. 171(4):1375 1380), high molecular weight melanomaantigen (HMW-MAA) (Natali et al. (1987) “Immunohistochemical DetectionOf Antigen In Human Primary And Metastatic Melanomas By The MonoclonalAntibody 140.240 And Its Possible Prognostic Significance,” Cancer 59:5563; Mittelman et al. (1990) “Active Specific Immunotherapy In PatientsWith Melanoma. A Clinical Trial With Mouse Antiidiotypic MonoclonalAntibodies Elicited With SyngeneicAnti-High-Molecular-Weight-Melanoma-Associated Antigen MonoclonalAntibodies,” J. Clin. Invest. 86:2136-2144), prostate specific membraneantigen, carcinoembryonic antigen (CEA) (Foon et al. (1995) “ImmuneResponse To The Carcinoembryonic Antigen In Patients Treated With AnAnti-Idiotype Antibody Vaccine,” J. Clin. Invest. 96(1):334-42),polymorphic epithelial mucin antigen, human milk fat globule antigen,Colorectal tumor-associated antigens such as: CEA, TAG-72 (Yokota et al.(1992) “Rapid Tumor Penetration Of A Single-Chain Fv And Comparison WithOther Immunoglobulin Forms,” Cancer Res. 52:3402-3408), CO17-1A(Ragnhammar et al. (1993) “Effect Of Monoclonal Antibody 17-1A AndGM-CSF In Patients With Advanced Colorectal Carcinoma—Long-Lasting,Complete Remissions Can Be Induced,” Int. J. Cancer 53:751-758); GICA19-9 (Herlyn et al. (1982) “Monoclonal Antibody Detection Of ACirculating Tumor-Associated Antigen. I. Presence Of Antigen In Sera OfPatients With Colorectal, Gastric, And Pancreatic Carcinoma,” J. Clin.Immunol. 2:135-140), CTA-1 and LEA, Burkitt's lymphoma antigen-38.13,CD19 (Ghetie et al. (1994) “Anti-CD19 Inhibits The Growth Of HumanB-Cell Tumor Lines In Vitro And Of Daudi Cells In SCID Mice By InducingCell Cycle Arrest,” Blood 83:1329-1336), human B-lymphoma antigen-CD20(Reff et al. (1994) “Depletion Of B Cells In Vivo By A Chimeric MouseHuman Monoclonal Antibody To CD20,” Blood 83:435-445), CD33 (Sgouros etal. (1993) “Modeling And Dosimetry Of Monoclonal Antibody M195(Anti-CD33) In Acute Myelogenous Leukemia,” J. Nucl. Med. 34:422-430),melanoma specific antigens such as ganglioside GD2 (Saleh et al. (1993)“Generation Of A Human Anti-Idiotypic Antibody That Mimics The GD2Antigen,” J. Immunol., 151, 3390-3398), ganglioside GD3 (Shitara et al.(1993) “A Mouse/Human Chimeric Anti-(Ganglioside GD3) Antibody WithEnhanced Antitumor Activities,” Cancer Immunol. Immunother. 36:373-380),ganglioside GM2 (Livingston et al. (1994) “Improved Survival In StageIII Melanoma Patients With GM2 Antibodies: A Randomized Trial OfAdjuvant Vaccination With GM2 Ganglioside,” J. Clin. Oncol.12:1036-1044), ganglioside GM3 (Hoon et al. (1993) “Molecular Cloning OfA Human Monoclonal Antibody Reactive To Ganglioside GM3 Antigen On HumanCancers,” Cancer Res. 53:5244-5250), tumor-specific transplantation typeof cell-surface antigen (TSTA) such as virally-induced tumor antigensincluding T-antigen DNA tumor viruses and envelope antigens of RNA tumorviruses, oncofetal antigen-alpha-fetoprotein such as CEA of colon,bladder tumor oncofetal antigen (Hellstrom et al. (1985) “MonoclonalAntibodies To Cell Surface Antigens Shared By Chemically Induced MouseBladder Carcinomas,” Cancer. Res. 45:2210-2188), differentiation antigensuch as human lung carcinoma antigen L6, L20 (Hellstrom et al. (1986)“Monoclonal Mouse Antibodies Raised Against Human Lung Carcinoma,”Cancer Res. 46:3917-3923), antigens of fibrosarcoma, human leukemia Tcell antigen-Gp37 (Bhattacharya-Chatterjee et al. (1988) “IdiotypeVaccines Against Human T Cell Leukemia. II. Generation AndCharacterization Of A Monoclonal Idiotype Cascade (Ab1, Ab2, and Ab3),”J. Immunol. 141(4):1398-1403), neoglycoprotein, sphingolipids, breastcancer antigen such as EGFR (Epidermal growth factor receptor), HER2antigen (p185^(HER2)), polymorphic epithelial mucin (PEM) (Hilkens etal. (1992) “Cell Membrane-Associated Mucins And TheirAdhesion-Modulating Property,” Trends in Biochem. Sci. 17:359-363),malignant human lymphocyte antigen-APO-1 (Trauth et al. (1989)“Monoclonal Antibody-Mediated Tumor Regression By Induction OfApoptosis,” Science 245:301-304), differentiation antigen (Feizi (1985)“Demonstration By Monoclonal Antibodies That Carbohydrate Structures OfGlycoproteins And Glycolipids Are Onco-Developmental Antigens,” Nature314:53-57) such as I antigen found in fetal erthrocytes and primaryendoderm, I(Ma) found in gastric adencarcinomas, M18 and M39 found inbreast epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, My1,VIM-D5, and D₁56-22 found in colorectal cancer, TRA-1-85 (blood groupH), C14 found in colonic adenocarcinoma, F3 found in lungadenocarcinoma, AH6 found in gastric cancer, Y hapten, Le^(y) found inembryonal carcinoma cells, TL5 (blood group A), EGF receptor found inA431 cells, E₁ series (blood group B) found in pancreatic cancer, FC10.2found in embryonal carcinoma cells, gastric adenocarcinoma, CO-514(blood group Le^(a)) found in adenocarcinoma, NS-10 found inadenocarcinomas, CO-43 (blood group Le^(b)), G49, EGF receptor, (bloodgroup ALe^(b)/Le^(y)) found in colonic adenocarcinoma, 19.9 found incolon cancer, gastric cancer mucins, T₅A₇ found in myeloid cells, R₂₄found in melanoma, 4.2, GD3, D1.1, OFA-1, GM2, OFA-2, GD2, M1:22:25:8found in embryonal carcinoma cells and SSEA-3, SSEA-4 found in 4-8-cellstage embryos. In another embodiment, the antigen is a T cell receptorderived peptide from a cutaneous T cell lymphoma (see Edelson (1998)“Cutaneous T-Cell Lymphoma: A Model For Selective Immunotherapy,” CancerJ Sci Am. 4:62-71).

The antibodies of the invention can be used in combination with anytherapeutic cancer antibodies known in the art to enhance the efficacyof treatment. For example, the antibodies of the invention can be usedwith any of the antibodies in Table 4, that have demonstratedtherapeutic utility in cancer treatment. The antibodies of the inventionenhance the efficacy of treatment of the therapeutic cancer antibodiesby enhancing at least one antibody-mediated effector function of saidtherapeutic cancer antibodies. In one particular embodiment, theantibodies enhance the efficacy of treatment by enhancing the complementdependent cascade of said therapeutic cancer antibodies. In anotherembodiment of the invention, the antibodies of the invention enhance theefficacy of treatment by enhancing the phagocytosis and opsonization ofthe targeted tumor cells. In another embodiment of the invention, theantibodies of the invention enhance the efficacy of treatment byenhancing antibody-dependent cell-mediated cytotoxicity (“ADCC”) indestruction of the targeted tumor cells.

Antibodies of the invention can also be used in combination withcytosine-guanine dinucleotides (“CpG”)-based products that have beendeveloped (Coley Pharmaceuticals) or are currently being developed asactivators of innate and acquired immune responses. For example, theinvention encompasses the use of CpG 7909, CpG 8916, CpG 8954 (ColeyPharmaceuticals) in the methods and compositions of the invention forthe treatment and/or prevention of cancer (See also Warren et al. (2002)“Synergism Between Cytosine-Guanine Oligodeoxynucleotides And MonoclonalAntibody In The Treatment Of Lymphoma,” Semin. Oncol., 29(1 Suppl 2):9397; Warren et al. (2000) “CpG Oligodeoxynucleotides Enhance MonoclonalAntibody Therapy Of A Murine Lymphoma,” Clin. Lymphoma, 1(1):57-61,which are incorporated herein by reference).

Antibodies of the invention can be used in combination with atherapeutic antibody that does not mediate its therapeutic effectthrough cell killing to potentiate the antibody's therapeutic activity.In a specific embodiment, the invention encompasses use of theantibodies of the invention in combination with a therapeutic apoptosisinducing antibody with agonisitc activity, e.g., an anti-Fas antibody.Anti-Fas antibodies are known in the art and include for example, Jo2(Ogasawara et al. (1993) “Lethal Effect Of The Anti-Fas Antibody InMice,” Nature 364: 806-809) and HFE7 (Ichikawa et al. (2000) “A NovelMurine Anti-Human Fas mAb Which Mitigates Lymphadenopathy WithoutHepatotoxicity,” Int. Immunol. 12: 555-562). Although not intending tobe bound by a particular mechanisms of action, FcγRIIB has beenimplicated in promoting anti-Fas mediated apoptosis, see, e.g., Xu etal. (2003) “FcRs Modulate Cytotoxicity Of Anti-Fas Antibodies:Implications For Agonistic Antibody-Based Therapeutics,” J. Immunol.171: 562-568. In fact the extracellular domain of FcγRIIB may serve as across-linking agent for Fas receptors, leading to a functional complexand promoting Fas dependent apoptosis. In some embodiments, theantibodies of the invention block the interaction of anti-Fas antibodiesand FcγRIIB, leading to a reduction in Fas-mediated apoptotic activity.Antibodies of the invention that result in a reduction in Fas-mediatedapoptotic activity are particularly useful in combination with anti-Fasantibodies that have undesirable side effects, e.g., hepatotoxicity. Inother embodiments, the antibodies of the invention enhance theinteraction of anti-Fas antibodies and FcγRIIB, leading to anenhancement of Fas-mediated apoptotic activity. Combination of theantibodies of the invention with therapeutic apoptosis inducingantibodies with agonisitc activity have an enhanced therapeuticefficacy.

Therapeutic apoptosis inducing antibodies used in the methods of theinvention may be specific for any death receptor known in the art forthe modulation of apoptotic pathway, e.g., TNFR receptor family.

The invention provides a method of treating diseases with impairedapoptotic mediated signaling, e.g., cancer. In a specific embodiment,the invention encompasses a method of treating a disease with deficientFas-mediated apoptosis, said method comprising administering an antibodyof the invention in combination with an anti-Fas antibody.

In some embodiments, the agonistic antibodies of the invention areparticularly useful for the treatment of tumors of non-hematopoieticorigin, including tumors of melanoma cells. Although not intending to bebound by a particular mechanism of action, the efficacy of the agonisticantibodies of the invention is due, in part, to activation of FcγRIIBinhibitory pathway, as tumors of non-hematopoietic origin, includingtumors of melanoma cells express FcγRIIB. Recent experiments have infact shown that expression of FcγRIIB in melanoma cells modulates tumorgrowth by direct interaction with anti-tumor antibodies (e.g., bybinding the Fc region of the anti-tumor antibodies) in anintracytoplasmic-dependent manner (Cassard et al. (2002) “Modulation OfTumor Growth By Inhibitory Fc(gamma) Receptor Expressed By HumanMelanoma Cells,” Journal of Clinical Investigation, 110(10): 1549-1557).

In some embodiments, the invention encompasses use of the antibodies ofthe invention in combination with therapeutic antibodies thatimmunospecifically bind to tumor antigens that are not expressed on thetumor cells themselves, but rather on the surrounding reactive and tumorsupporting, non-malignant cells comprising the tumor stroma. The tumorstroma comprises endothelial cells forming new blood vessels and stromalfibroblasts surrounding the tumor vasculature. In a specific embodiment,an antibody of the invention is used in combination with an antibodythat immunospecifically binds a tumor antigen on an endothelial cell. Ina preferred embodiment, an antibody of the invention is used incombination with an antibody that immunospecifically binds a tumorantigen on a fibroblast cell, e.g., fibroblast activation protein (FAP).FAP is a 95 KDa homodimeric type II glycoprotein which is highlyexpressed in stromal fibroblasts of many solid tumors, including, butnot limited to lung, breast, and colorectal carcinomas. (See, e.g.,Scanlan et al. (1994) “Molecular Cloning Of Fibroblast ActivationProtein Alpha, A Member Of The Serine Protease Family SelectivelyExpressed In Stromal Fibroblasts Of Epithelial Cancers,” Proc. Natl.Acad. USA, 91: 5657-5661; Park et al. (1999) “Fibroblast ActivationProtein, A Dual Specificity Serine Protease Expressed In Reactive HumanTumor Stromal Fibroblasts,” J. Biol. Chem., 274: 36505-36512; Rettig etal. (1988) “Cell-Surface Glycoproteins Of Human Sarcomas: DifferentialExpression In Normal And Malignant Tissues And Cultured Cells,” Proc.Natl. Acad. Sci. USA 85: 3110-3114; Garin-Chesa et al. (1990) “CellSurface Glycoprotein Of Reactive Stromal Fibroblasts As A PotentialAntibody Target In Human Epithelial Cancers,” Proc. Natl. Acad. Sci. USA87: 7235-7239). Antibodies that immunospecifically bind FAP are known inthe art and encompassed within the invention, see, e.g., Wuest et al.(2001) “Construction Of A Bispecific Single Chain Antibody ForRecruitment Of Cytotoxic T Cells To The Tumour Stroma Associated AntigenFibroblast Activation Protein,” Journal of Biotechnology, 159-168;Mersmann et al. (2001) “Human Antibody Derivatives Against TheFibroblast Activation Protein For Tumor Stroma Targeting Of Carcinomas,”Int. J. Cancer, 92: 240-248; U.S. Pat. No. 6,455,677; all of which areincorporated herein in by reference in their entireties.

Recently IgEs have been implicated as mediators of tumor growth and infact IgE-targeted immediate hypersensitivity and allergic inflammationreactions have been proposed as possible natural mechanisms involved inanti-tumor responses (For a review see, e.g., Vena et al. (1992)“Allergy-Related Diseases And Cancer: An Inverse Association,” Am.Journal of Epidemiol. 122: 66-74; Eriksson et al. (1995) “A ProspectiveStudy Of Cancer Incidence In A Cohort Examined For Allergy,” Allergy 50:718-722). In fact a recent study has shown loading tumor cells with IgEsreduces tumor growth, leading in some instances to tumor rejection.According to the study, IgE loaded tumor cells not only possess atherapeutic potential but also confer long term antitumor immunity,including activation of innate immunity effector mechanism and T-cellmediated adaptive immune response, see Reali et al. (2001) “IgEsTargeted On Tumor Cells: Therapeutic Activity And Potential In TheDesign Of Tumor Vaccines,” Cancer Res. 61: 5517-5522; which isincorporated herein by reference in its entirety. The antagonisticantibodies of the invention may be used in the treatment and/orprevention of cancer in combination with administration of IgEs in orderto enhance the efficacy of IgE-mediated cancer therapy. Although notintending to be bound by a particular mechanism of action the antibodiesof the invention enhance the therapeutic efficacy of IgE treatment oftumors, by blocking the inhibitory pathway. The antagonistic antibodiesof the invention may enhance the therapeutic efficacy of IgE mediatedcancer therapy by (i.) enhancing the delay in tumor growth; (ii.)enhancing the decrease in the rate of tumor progression; (iii.)enhancing tumor rejection; or (iv.) enhancing protective immune relativeto treatment of cancer with IgE alone.

Cancer therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in theliterature, see, e.g., Physician's Desk Reference (56^(th) ed., 2002,which is incorporated herein by reference).

B. B Cell Malignancies

The agonistic antibodies of the invention are useful for treating orpreventing any B cell malignancies, particularly non-Hodgkin's lymphomaand chronic lymphocytic leukemia. FcγRIIB, is a target for deregulationby chromosomal translocation in malignant lymphoma, particularly inB-cell non-Hodgkin's lymphoma (See Callanan et al. (2000) “The IgG FcReceptor, FcgammaRIIB, Is A Target For Deregulation By ChromosomalTranslocation In Malignant Lymphoma,” Proc. Natl. Acad. Sci. U.S.A.,97(1):309-314). Thus, the antibodies of the invention are useful fortreating or preventing any chronic lymphocytic leukemia of the B celllineage. Chronic lymphocytic leukemia of the B cell lineage are reviewedby Freedman (See review by Freedman (1990) “Immunobiology Of ChronicLymphocytic Leukemia,” Hemtaol. Oncol. Clin. North Am. 4:405-429).Although not intending to be bound by any mechanism of action, theagonistic antibodies of the invention inhibit or prevent B cellmalignancies inhibiting B cell proliferation and/or activation. Theinvention also encompasses the use of the agonistic antibodies of theinvention in combination with other therapies known (e.g., chemotherapyand radiotherapy) in the art for the prevention and/or treatment of Bcell malignancies. The invention also encompasses the use of theagonistic antibodies of the invention in combination with otherantibodies known in the art for the treatment and or prevention ofB-cell malignancies. For example, the agonistic antibodies of theinvention can be used in combination with the anti-C22 or anti-CD19antibodies disclosed by Goldenberg et al. (U.S. Pat. No. 6,306,393).

Antibodies of the invention can also be used in combination with forexample but not by way of limitation, ONCOSCINT® (¹¹¹In-satumomabpantedide) (target: CEA), VERLUMA® (⁹⁹Tc-nofetumomab merpentan) (target:GP40), PROSTASCINT® (capromab pentedide) (target: PSMA), CEA-SCAN®(arcitumomab) (target: CEA), RITUXAN® (rituximab) (target: CD20),HERCEPTIN® (trastuzumab) (target: HER-2), CAMPATH® (alemtuzumab (target:CD52), MYLOTARG® (gemtuzumab ozogamicin) (target: CD33), and ZEVALIN®(ibritumomab tiuxetan) (target: CD20).

C. Allergy

The invention provides methods for treating or preventing anIgE-mediated and or FcεRI mediated allergic disorder in a subject inneed thereof, comprising administering to said subject a therapeuticallyeffective amount of the agonistic antibodies or fragments thereof of theinvention. Although not intending to be bound by a particular mechanismof action, antibodies of the invention are useful in inhibitingFcεRI-induced mast cell activation, which contributes to acute and latephase allergic responses (Metcalfe et al. (1997) “Mast Cells,” Physiol.Rev. 77:1033-1079). Preferably, the agonistic antibodies of theinvention have enhanced therapeutic efficacy and/or reduced side effectsin comparison with the conventional methods used in the art for thetreatment and/or prevention of IgE mediated allergic disorders.Conventional methods for the treatment and/or prevention of IgE mediatedallergic disorders include, but are not limited to, anti-inflammatorydrugs (e.g., oral and inhaled corticosteroids for asthma),antihistamines (e.g., for allergic rhinitis and atopic dermatitis),cysteinyl leukotrienes (e.g., for the treatment of asthma); anti-IgEantibodies; and specific immunotherapy or desensitization.

Examples of IgE-mediated allergic responses include, but are not limitedto, asthma, allergic rhinitis, gastrointestinal allergies, eosinophilia,conjunctivitis, atopic dermatitis, urticaria, anaphylaxis, or golmerularnephritis.

The invention encompasses molecules, e.g., immunoglobulins, engineeredto form complexes with FcεRI and human FcγRIIB, i.e., specifically bindFcεRI and human FcγRIIB. Preferably, such molecules have therapeuticefficacy in IgE and FcεRI-mediated disorders. Although not intending tobe bound by a particular mechanism of action, the therapeutic efficacyof these engineered molecules is, in part, due to their ability toinhibit mast cell and basophil function.

In a specific embodiment, molecules that specifically bind FcεRI andhuman FcγRIIB are chimeric fusion proteins comprising a binding site forFcεRI and a binding site for FcγRIIB. Such molecules may be engineeredin accordance with standard recombinant DNA methodologies known to oneskilled in the art. In a preferred specific embodiment, a chimericfusion protein for use in the methods of the invention comprises anF(ab′) single chain of an anti-FcγRIIB monoclonal antibody of theinvention fused to a region used as a bridge to link the huFcε to theC-terminal region of the F(ab′) single chain of the anti-FcγRIIBmonoclonal antibody. One exemplary chimeric fusion protein for use inthe methods of the invention comprises the following: V_(L)/C_(H)(FcγRIIB)-hinge-V_(H)/C_(H) (FcγRIIB)-LINKER-C_(H)ε2-C_(H)ε3-C_(H)ε4.The linker for the chimeric molecules may be five, ten, preferablyfifteen amino acids in length. The length of the linker may vary toprovide optimal binding of the molecule to both FcγRIIB and FcεRI. In aspecific embodiment, the linker is a 15 amino acid linker, consisting ofthe sequence: (Gly₄Ser)₃. Although not intending to be bound by aparticular mechanism of action, the flexible peptide linker facilitateschain pairing and minimizes possible refolding and it will also allowthe chimeric molecule to reach the two receptors, i.e., FcγRIIB andFcεRI on the cells and cross-link them. Preferably, the chimericmolecule is cloned into a mammalian expression vector, e.g., pCI-neo,with a compatible promoter, e.g., cytomegalovirus promoter. The fusionprotein prepared in accordance with the methods of the invention willcontain the binding site for FcεRI (CHε2CHε3) and for FcγRIIB(VL/CL,-hinge-VH/CH). The nucleic acid encoding the fusion proteinprepared in accordance with the methods of the invention is preferablytransfected into 293 cells and the secreted protein is purified usingcommon methods known in the art.

Binding of the chimeric molecules to both human FcεRI and FcγRIIB may beassessed using common methods known to one skilled in the art fordetermining binding to an FcγR. Preferably, the chimeric molecules ofthe invention have therapeutic efficacy in treating IgE mediateddisorders, for example, by inhibiting antigen-driven degranulation andinhibition of cell activation. The efficacy of the chimeric molecules ofthe invention in blocking IgE driven FcεRI-mediated mast celldegranulation may be determined in transgenic mice, which have beenengineered to express the human FcεRα and human FcγRIIB, prior to theiruse in humans.

The invention provides the use of bispecific antibodies for thetreatment and/or prevention of IgE-mediated and/or FcεRI-mediatedallergic disorders. A bispecific antibody (BsAb) binds to two differentepitopes usually on distinct antigens. BsAbs have potential clinicalutility and they have been used to target viruses, virally infectedcells and bacterial pathogens as well as to deliver thrombolitic agentsto blood clots (Cao (1998) “Bispecific Antibodies As NovelBioconjugates,” Bioconj. Chem. 9: 635-644; Koelemij et al. (1999)“Bispecific Antibodies In Cancer Therapy, From The Laboratory To TheClinic,” J. Immunother., 22, 514-524; Segal et al. (1999) “BispecificAntibodies In Cancer Therapy,” Curr. Opin. Immunol., 11, 558-562). Thetechnology for the production of BsIgG and other related bispecificmolecules is available (see, e.g., Carter et al. (2001) “BispecificHuman IgG By Design,” J. of Immunol. Methods, 248, 7-15; Segal et al.(2001) “Introduction: Bispecific Antibodies,” J. of Immunol. Methods,248, 71-76, which are incorporated herein by reference in theirentirety). The instant invention provides bispecific antibodiescontaining one F(ab′) of the anti-FcγRIIB antibody and one F(ab′) of anavailable monoclonal anti-hulgE antibody which aggregates two receptors,FcγRIIB and FcεRI, on the surface of the same cell. Any methodologyknown in the art and disclosed herein may be employed to generatebispecific antibodies for use in the methods of the invention. In aspecific embodiment, the BsAbs will be produced by chemicallycross-linking F(ab′) fragments of an anti-FcγRIIB antibody and ananti-hulge antibody as described previously, see, e.g., Glennie et al.,1995, Tumor Immunobiology, Oxford University press, Oxford, p. 225;which is incorporated herein by reference in its entirety). The F(ab′)fragments may be produced by limited proteolysis with pepsin and reducedwith mercaptoethanol amine to provide Fab′ fragments with freehinge-region sulfhydryl (SH) groups. The SH group on one of the Fab′(SH) fragments may be alkylated with excess 0-phenylenedimaleimide(0-PDM) to provide a free maleimide group (mal). The two preparationsFab′(mal) and Fab′(SH) may be combined at an appropriate ratio,preferably 1:1 to generate heterodimeric constructs. The BsAbs can bepurified by size exclusion chromatography and characterized by HPLCusing methods known to one skilled in thr art.

In particular, the invention encompasses bispecific antibodiescomprising a first heavy chain-light chain pair that binds FcγRIIB withgreater affinity than said heavy chain-light chain pair binds FcγRIIA,and a second heavy chain-light chain pair that binds IgE receptor, withthe provision that said first heavy chain-light chain pair binds FcγRIIBfirst. The bispecific antibodies of the invention can be engineeredusing standard techniques known in the art to ensure that the binding toFcγRIIB precedes the binding to the IgE receptor. It will be understoodby one skilled in the art that it is possible to engineer the bispecificantibodies, for example, such that said bispecific antibodies bindFcγRIIB with greater affinity than said antibodies bind IgE receptor.Additionally, the bispecific antibodies can be engineered by techniquesknown in the art, such that the hinge size of the antibody can beincreased in length, for example, by adding linkers, to provide thebispecific antibodies with flexibility to bind the IgE receptor andFcγRIIB receptor on the same cell.

The antibodies of the invention can also be used in combination withother therapeutic antibodies or drugs known in the art for the treatmentor prevention of IgE-mediated allergic disorders. For example, theantibodies of the invention can be used in combination with any of thefollowing: azelastine (ASTELIN®), beclomethasone dipropionate inhaler(VANCERIL®), beclomethasone dipropionate nasal inhaler/spray(VANCENASE®), Beconase budesonide nasal inhaler/spray (RHINOCORT®),cetirizine (ZYRTEC®), chlorpheniramine, pseudoephedrine, Deconamine(SUDAFED®), cromolyn (NASALCROM®, INTAL®, OPTICROM®), desloratadine(CLARINEX®), fexofenadine and pseudoephedrine (ALEGRA-D®), fexofenadine(ALLEGRA®), flunisolide nasal spray (NASALIDE®), fluticasone propionatenasal inhaler/spray (FLONASE®), fluticasone propionate oral inhaler(FLOVENT®), hydroxyzine (VISTARIL®, ATARAX®), loratadine,pseudoephedrine (CLARITIN-D®), loratadine (CLARITIN®), prednisolone(PREDNISOLONE®, PEDIAPRED® Oral Liquid, MEDROL® prednisone, DELTASONE®,Liquid Predsalmeterol), salmeterol xinafoate (SEREVENT®), triamcinoloneacetonide inhaler (AZMACORT®), triamcinolone acetonide nasalinhaler/spray (NASACORT®, or NASACORT AQ®). Antibodies of the inventioncan be used in combination with cytosine-guanine dinucleotides(“CpG”)-based products that have been developed (Coley Pharmaceuticals)or are currently being developed as activators of innate and acquiredimmune responses. For example, the invention encompasses the use of CpG7909, CpG 8916, CpG 8954 (Coley Pharmaceuticals) in the methods andcompositions of the invention for the treatment and/or prevention ofIgE-mediated allergic disorders (See also Weeratna et al. (2001) “CpGODN Can Re-Direct The Th Bias Of Established Th2 Immune Responses InAdult And Young Mice,” FEMS Immunol Med. Microbiol., 32(1):65-71, whichis incorporated herein by reference).

The invention encompasses the use of the antibodies of the invention incombination with any therapeutic antibodies known in the art for thetreatment of allergy disorders, e.g., XOLAIR™ (Omalizumab; Genentech);rhuMAB-E25 (BioWorld Today, Nov. 10, 1998, p. 1; Genentech); CGP-51901(humanized anti-IgE antibody), etc.

Additionally, the invention encompasses the use of the antibodies of theinvention in combination with other compositions known in the art forthe treatment of allergy disorders. In particular, methods andcompositions disclosed in Carson et al. (U.S. Pat. No. 6,426,336; US2002/0035109 A1; US 2002/0010343) is incorporated herein by reference inits entirety.

D. Anti-Cancer Agents and Therapeutic Antibodies

In a specific embodiment, the methods of the invention encompass theadministration of one or more angiogenesis inhibitors such as but notlimited to: Angiostatin (plasminogen fragment); antiangiogenicantithrombin III; Angiozyme; ABT-627; Bay 12-9566; Benefin; Bevacizumab;BMS-275291; cartilage-derived inhibitor (CDI); CAI; CD59 complementfragment; CEP-7055; Col 3; Combretastatin A-4; Endostatin (collagenXVIII fragment); Fibronectin fragment; Gro-beta; Halofuginone;Heparinases; Heparin hexasaccharide fragment; HMV833; Human chorionicgonadotropin (hCG); IM-862; Interferon alpha/beta/gamma; Interferoninducible protein (IP-10); Interleukin-12; Kringle 5 (plasminogenfragment); Marimastat; Metalloproteinase inhibitors (TIMPs);2-Methoxyestradiol; MMI 270 (CGS 27023A); MoAb IMC-1C11; Neovastat;NM-3; Panzem; PI-88; Placental ribonuclease inhibitor; Plasminogenactivator inhibitor; Platelet factor-4 (PF4); Prinomastat; Prolactin 16kD fragment; Proliferin-related protein (PRP); PTK 787/ZK 222594;Retinoids; Solimastat; Squalamine; SS 3304; SU 5416; SU6668; SU11248;Tetrahydrocortisol-S; tetrathiomolybdate; thalidomide; Thrombospondin-1(TSP-1); TNP-470; Transforming growth factor-beta (TGF-b);Vasculostatin; Vasostatin (calreticulin fragment); ZD6126; ZD 6474;farnesyl transferase inhibitors (FTI); and bisphosphonates.

Anti-cancer agents that can be used in combination with antibodies ofthe invention in the various embodiments of the invention, includingpharmaceutical compositions and dosage forms and kits of the invention,include, but are not limited to: acivicin; aclarubicin; acodazolehydrochloride; acronine; adozelesin; aldesleukin; altretamine;ambomycin; ametantrone acetate; aminoglutethimide; amsacrine;anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa;azotomycin; batimastat; benzodepa; bicalutamide; bisantrenehydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate;brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone;caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine;dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;estramustine; estramustine phosphate sodium; etanidazole; etoposide;etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine;fenretinide; floxuridine; fludarabine phosphate; fluorouracil;fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabinehydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; interleukin II (including recombinant interleukin II, orrIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1;interferon alfa-n3; interferon beta-I a; interferon gamma-I b;iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride. Other anti-cancer drugs include,but are not limited to: 20-epi-1,25 dihydroxyvitamin D3;5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel;docosanol; dolasetron; doxifluridine; droloxifene; dronabinol;duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;eflornithine; elemene; emitefur; epirubicin; epristeride; estramustineanalogue; estrogen agonists; estrogen antagonists; etanidazole;etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide;filgrastim; finasteride; flavopiridol; flezelastine; fluasterone;fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane;fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathioneinhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin;ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;ilomastat; imidazoacridones; imiquimod; immunostimulant peptides;insulin-like growth factor-1 receptor inhibitor; interferon agonists;interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-;iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofuran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer. Preferred additional anti-cancer drugs are 5-fluorouraciland leucovorin.

Examples of therapeutic antibodies that can be used in methods of theinvention include but are not limited to HERCEPTIN® (Trastuzumab)(Genentech, CA) which is a humanized anti-HER2 monoclonal antibody forthe treatment of patients with metastatic breast cancer; REOPRO®(abciximab) (Centocor) which is an anti-glycoprotein IIb/IIIa receptoron the platelets for the prevention of clot formation; ZENAPAX®(daclizumab) (Roche Pharmaceuticals, Switzerland) which is animmunosuppressive, humanized anti-CD25 monoclonal antibody for theprevention of acute renal allograft rejection; PANOREX® (edrecolomab)which is a murine anti-17-IA cell surface antigen IgG2a antibody (GlaxoWellcome/Centocor); BEC2 which is a murine anti-idiotype (GD3 epitope)IgG antibody (ImClone System); IMC-C225 which is a chimeric anti-EGFRIgG antibody (ImClone System); VITAXIN™ which is a humanized anti-αVβ3integrin antibody (Applied Molecular Evolution/MedImmune); CAMPATH®(alemtuzumab) 1H/LDP-03 which is a humanized anti CD52 IgG1 antibody(Leukosite); Smart M195 which is a humanized anti-CD33 IgG antibody(Protein Design Lab/Kanebo); RITUXAN® (rituximab) which is a chimericanti-CD20 IgG1 antibody (IDEC Pharm/Genentech, Roche/Zettyaku);LYMPHOCIDE® (epratuzumab) which is a humanized anti-CD22 IgG antibody(Immunomedics); ICM3 is a humanized anti-ICAM3 antibody (ICOS Pharm);IDEC-114 is a primatied anti-CD80 antibody (IDEC Pharm/Mitsubishi);ZEVALIN™ is a radiolabelled murine anti-CD20 antibody (IDEC/ScheringAG); IDEC-131 is a humanized anti-CD40L antibody (IDEC/Eisai); IDEC-151is a primatized anti-CD4 antibody (IDEC); IDEC-152 is a primatizedanti-CD23 antibody (IDEC/Seikagaku); SMART anti-CD3 is a humanizedanti-CD3 IgG (Protein Design Lab); 5G1.1 is a humanized anti-complementfactor 5 (C5) antibody (Alexion Pharm); D2E7 is a humanized anti-TNF-αantibody (CAT/BASF); CDP870 is a humanized anti-TNF-α Fab fragment(Celltech); IDEC-151 is a primatized anti-CD4 IgG1 antibody (IDECPharm/SmithKline Beecham); MDX-CD4 is a human anti-CD4 IgG antibody(Medarex/Eisai/Genmab); CDP571 is a humanized anti-TNF-α IgG4 antibody(Celltech); LDP-02 is a humanized anti-α4β7 antibody(LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4 IgGantibody (Ortho Biotech); ANTOVA® (ruplizumab) is a humanized anti-CD40LIgG antibody (Biogen); ANTEGREN® (natalizumab) is a humanized anti-IgGantibody (Elan); and CAT-152 is a human anti-TGF-β₂ antibody (CambridgeAb Tech).

Other examples of therapeutic antibodies that can be used in combinationwith the antibodies of the invention are presented in Table 4.

TABLE 4 Monoclonal Antibodies For Cancer Therapy That Can Be Used InCombination With The Antibodies Of The Invention Product Disease TargetABX-EGF Cancer EGF receptor OvaRex ovarian cancer tumor antigen CA125BravaRex metastatic cancers tumor antigen MUC1 Theragyn ovarian cancerPEM antigen (pemtumomabytrrium-90) Therex breast cancer PEM antigenblvatuzumab head & neck CD44 cancer Panorex Colorectal cancer 17-1AReoPro PTCA gp IIIb/IIIa ReoPro Acute MI gp IIIb/IIIa ReoPro Ischemicstroke gp IIIb/IIIa Bexocar NHL CD20 MAb, idiotypic 105AD7 colorectalcancer gp72 vaccine Anti-EpCAM cancer Ep-CAM MAb, lung cancer non-smallcell NA lung cancer Herceptin metastatic breast HER-2 cancer Herceptinearly stage breast HER-2 cancer Rituxan Relapsed/refractory CD20low-grade or follicular NHL Rituxan intermediate & CD20 high-grade NHLMAb-VEGF NSCLC, VEGF metastatic MAb-VEGF Colorectal cancer, VEGFmetastatic AMD Fab age-related CD18 macular degeneration E-26 (2^(nd)gen. IgE) allergic asthma & IgE rhinitis Zevalin (Rituxan + low grade ofCD20 yttrium-90) follicular, relapsed or refractory, CD20-positive, B-cell NHL and Rituximab- refractory NHL Cetuximab + innotecan refractoryEGF receptor colorectal carcinoma Cetuximab + cisplatin & newlydiagnosed EGF receptor radiation or recurrent head & neck cancerCetuximab + gemcitabine newly diagnosed EGF receptor metastaticpancreatic carcinoma Cetuximab + cisplatin + recurrent or EGF receptor5FU or Taxol metastatic head & neck cancer Cetuximab + carboplatin +newly diagnosed EGF receptor paclitaxel non-small cell lung carcinomaCetuximab + cisplatin head & neck EGF receptor cancer (extensiveincurable local- regional disease & distant metasteses) Cetuximab +radiation locally advanced EGF receptor head & neck carcinoma BEC2 +Bacillus Calmette small cell lung mimics ganglioside Guerin carcinomaGD3 BEC2 + Bacillus Calmette melanoma mimics ganglioside Guerin GD3IMC-1C11 colorectal cancer VEGF-receptor with liver metastesesnuC242-DM1 Colorectal, gastric, nuC242 and pancreatic cancer LymphoCideNon-Hodgkins CD22 lymphoma LymphoCide Y-90 Non-Hodgkins CD22 lymphomaCEA-Cide metastatic solid CEA tumors CEA-Cide Y-90 metastatic solid CEAtumors CEA-Scan (Tc-99m- colorectal cancer CEA labeled arcitumomab)(radioimaging) CEA-Scan (Tc-99m- Breast cancer CEA labeled arcitumomab)(radioimaging) CEA-Scan (Tc-99m- lung cancer CEA labeled arcitumomab)(radioimaging) CEA-Scan (Tc-99m- intraoperative CEA labeled arcitumomab)tumors (radio imaging) LeukoScan (Tc-99m- soft tissue CEA labeledsulesomab) infection (radioimaging) LymphoScan (Tc-99m- lymphomas CD22labeled) (radioimaging) AFP-Scan (Tc-99m- liver 7 gem-cell AFP labeled)cancers (radioimaging) HumaRAD-HN head & neck NA (+yttrium-90) cancerHumaSPECT colorectal imaging NA MDX-101 (CTLA-4) Prostate and otherCTLA-4 cancers MDX-210 (her-2 Prostate cancer HER-2 overexpression)MDX-210/MAK Cancer HER-2 Vitaxin Cancer αvβ₃ MAb 425 Various cancers EGFreceptor IS-IL-2 Various cancers Ep-CAM Campath (alemtuzumab) chronicCD52 lymphocytic leukemia CD20-streptavidin Non-Hodgkins CD20(+biotin-yttrium 90) lymphoma Avidicin (albumin + metastatic cancer NANRLU13) Oncolym (+iodine-131) Non-Hodgkins HLA-DR 10 beta lymphomaCotara (+iodine-131) unresectable DNA-associated malignant gliomaproteins C215 (+staphylococcal pancreatic cancer NA enterotoxin) MAb,lung/kidney cancer lung & kidney NA cancer nacolomab tafenatox colon &pancreatic NA (C242 + staphylococcal cancer enterotoxin) Nuvion T cellCD3 malignancies SMART M195 AML CD33 SMART 1D10 NHL HLA-DR antigenCEAVac colorectal cancer, CEA advanced TriGem metastatic GD2-gangliosidemelanoma & small cell lung cancer TriAb metastatic breast MUC-1 cancerCEAVac colorectal cancer, CEA advanced TriGem metastatic GD2-gangliosidemelanoma & small cell lung cancer TriAb metastatic breast MUC-1 cancerNovoMAb-G2 Non-Hodgkins NA radiolabeled lymphoma Monopharm C colorectal& SK-1 antigen pancreatic carcinoma GlioMAb-H (+gelonin gliorna,melanoma NA toxin) & neuroblastoma Rituxan Relapsed/refractory CD20low-grade or follicular NHL Rituxan intermediate & CD20 high-grade NHLING-1 adenomcarcinoma Ep-CAM

VII. Vaccine Therapy

The invention provides a method for enhancing an immune response to avaccine composition in a subject, said method comprising administeringto said subject an antibody or a fragment thereof that specificallybinds FcγRIIB with greater affinity than said antibody or a fragmentthereof binds FcγRIIA, and a vaccine composition, wherein said antibodyor a fragment thereof enhances the immune response to said vaccinecomposition. In one particular embodiment, said antibody or a fragmentthereof enhances the immune response to said vaccine composition byenhancing antigen presentation/and or antigen processing of the antigento which the vaccine is directed at. Any vaccine composition known inthe art is useful in combination with the antibodies or fragmentsthereof of the invention.

In one embodiment, the invention encompasses the use of the antibodiesof the invention in combination with any cancer vaccine known in theart, e.g., CANVAXIN™ (Cancer Vax, Corporation, melanoma and coloncancer); ONCOPHAGE® (HSPPC-96; Antigenics; metastatic melanoma);HER-2/neu cancer vaccine, etc. The cancer vaccines used in the methodsand compositions of the invention can be, for example, antigen-specificvaccines, anti-idiotypic vaccines, dendritic cell vaccines, or DNAvaccines. The invention encompasses the use of the antibodies of theinvention with cell-based vaccines as described by Segal et al. (U.S.Pat. No. 6,403,080), which is incorporated herein by reference in itsentirety. The cell-based vaccines used in combination with theantibodies of the invention can be either autologous or allogeneic.Briefly, the cancer-based vaccines as described by Segal et al. arebased on Opsonokine™ product by Genitrix, LLC. Opsonokines™ aregenetically engineered cytokines that, when mixed with tumor cells,automatically attach to the surface of the cells. When the “decorated”cells are administered as a vaccine, the cytokine on the cells activatescritical antigen presenting cells in the recipient, while also allowingthe antigen presenting cells to ingest the tumor cells. The antigenpresenting cells are then able to instruct “killer” T cells to find anddestroy similar tumor cells throughout the body. Thus, the Opsonokine™product converts the tumor cells into a potent anti-tumorimmunotherapeutic.

In one embodiment, the invention encompasses the use of the antibodiesof the invention in combination with any allergy vaccine known in theart. The antibodies of the invention, can be used, for example, incombination with recombinant hybrid molecules coding for the majortimothy grass pollen allergens used for vaccination against grass pollenallergy, as described by Linhart et al. (2002) “Combination Vaccines ForThe Treatment Of Grass Pollen Allergy Consisting Of GeneticallyEngineered Hybrid Molecules With Increased Immunogenicity,” FASEBJournal, 16(10):1301-1303, which is incorporated herein by reference inits entirety. In addition, the antibodies of the invention can be usedin combination with DNA-based vaccinations described by Horner et al.(2002) “Immunostimulatory Dna-Based Therapeutics For Experimental AndClinical Allergy,” Allergy, 57 Suppl, 72:24-29. Antibodies of theinvention can be used in combination with Bacille Clamett-Guerin (“BCG”)vaccination as described by Choi et al. (2002) “Therapeutic Effects OfBcg Vaccination In Adult Asthmatic Patients: A Randomized, ControlledTrial,” Ann. Allergy Asthma Immunology, 88(6): 584-591); and Barlan etal. (2002) “The Impact Of In Vivo Calmette-Guerin BacillusAdministration On In Vitro Ige Secretion In Atopic Children,” JournalAsthma, 39(3):239-246, both of which are incorporated herein byreference in entirety, to downregulate IgE secretion. The antibodies ofthe invention are useful in treating food allergies. In particular, theantibodies of the invention can be used in combination with vaccines orother immunotherapies known in the art (see Hourihane et al. (2002)“Recent Advances In Peanut Allergy,” Curr. Opin. Allergy Clin. Immunol.2(3):227-231) for the treatment of peanut allergies

The methods and compositions of the invention can be used in combinationwith vaccines, in which immunity for the antigen(s) is desired. Suchantigens may be any antigen known in the art. The antibodies of theinvention can be used to enhance an immune response, for example, toinfectious agents, diseased or abnormal cells such as, but not limitedto, bacteria (e.g., gram positive bacteria, gram negative bacteria,aerobic bacteria, Spirochetes, Mycobacteria, Rickettsias, Chlamydias,etc.), parasites, fungi (e.g., Candida albicans, Aspergillus, etc.),viruses (e.g., DNA viruses, RNA viruses, etc.), or tumors. Viralinfections include, but are not limited to, human immunodeficiency virus(HIV); hepatitis A virus, hepatitis B virus, hepatitis C virus,hepatitis D virus, or other hepatitis viruses; cytomagaloviruses, herpessimplex virus-1 (-2, -3, -4, -5, -6), human papilloma viruses;Respiratory syncytial virus (RSV), Parainfluenza virus (PIV), EpsteinBarr virus, or any other viral infections.

The invention encompasses the use of the antibodies of the invention toenhance a humoral and/or cell mediated response against the antigen(s)of the vaccine composition. The invention further encompasses the use ofthe antibodies of the invention to either prevent or treat a particulardisorder, where an enhanced immune response against a particular antigenor antigens is effective to treat or prevent the disease or disorder.Such diseases and disorders include, but are not limited to, viralinfections, such as HIV, CMV, hepatitis, herpes virus, measles, etc.,bacterial infections, fungal and parasitic infections, cancers, and anyother disease or disorder amenable to treatment or prevention byenhancing an immune response against a particular antigen or antigens.

VIII. Breaking Tolerance to an Antigen

Certain cancers may be associated with an ability of the tumors tocircumvent an immune response against their antigens, i.e., tolerance tothese antigens exists. (See Mapara et al. (2004) “Tolerance and Cancer:Mechanisms Of Tumor Evasion and Strategies For Breaking Tolerance,” J.Clin. Oncol. 22:1136-1151.) Accordingly, a goal in tumor immunotherapyis to break tolerance to tumor antigens in order to induce an antitumorresponse. Eliciting an immune response against a foreign antigen that isotherwise recognized by the host as a “self” antigen breaks tolerance tothat antigen.

Thus, in certain embodiments, the invention provides a method forbreaking tolerance to an antigen in a patient by administering to apatient in need thereof (i.) an antigen-antibody complex comprising theantigen and (ii.) an antibody or fragment thereof that specificallybinds the extracellular domain of human FcγRIIB and blocks the Fcbinding site of human FcγRIIB, thereby breaking tolerance in saidpatient to the antigen. The antibody or fragment thereof can beadministered before, concurrently with, or after administration of saidantigen-antibody complex.

Antigen-presenting cells, such as dendritic cells, coexpress activatingand inhibitory Fc gamma receptors. Without being bound by theory, whenantibodies that block Fc binding to FcγRIIB are present, theantigen-antibody complexes comprising an antigen are primarily taken upby non-inhibitory receptors on antigen-presenting cells elicting animmune response to the antigen.

In certain embodiments, the antigen is an antigen that is associatedwith a cancer or a neoplastic disease. In another aspect, the antigen isspecific to a cancer cell or a neoplastic cell. The antigen can also bean antigen of a pathogen, such as, e.g., a virus, a bacterium, or aprotozoa. Representative antigens have been disclosed herein.

IX. Compositions and Methods of Administering

The invention provides methods and pharmaceutical compositionscomprising antibodies of the invention. The invention also providesmethods of treatment, prophylaxis, and amelioration of one or moresymptoms associated with a disease, disorder or infection byadministering to a subject an effective amount of a fusion protein or aconjugated molecule of the invention, or a pharmaceutical compositioncomprising a fusion protein or conjugated molecules of the invention. Ina preferred aspect, an antibody or fusion protein or conjugatedmolecule, is substantially purified (i.e., substantially free fromsubstances that limit its effect or produce undesired side-effects). Ina specific embodiment, the subject is an animal, preferably a mammalsuch as non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.)and a primate (e.g., monkey such as, a cynomolgous monkey and a human).In a preferred embodiment, the subject is a human.

Various delivery systems are known and can be used to administer acomposition comprising antibodies of the invention, e.g., encapsulationin liposomes, microparticles, microcapsules, recombinant cells capableof expressing the antibody or fusion protein, receptor-mediatedendocytosis (See, e.g., Wu et al. (1987) “Receptor-Mediated In VitroGene Transformation By A Soluble DNA Carrier System,” J. Biol. Chem.262:4429-4432), construction of a nucleic acid as part of a retroviralor other vector, etc.

In some embodiments, the antibodies of the invention are formulated inliposomes for targeted delivery of the antibodies of the invention.Liposomes are vesicles comprised of concentrically ordered phopsholipidbilayers that encapsulate an aqueous phase. Liposomes typically comprisevarious types of lipids, phospholipids, and/or surfactants. Thecomponents of liposomes are arranged in a bilayer configuration, similarto the lipid arrangement of biological membranes. Liposomes areparticularly preferred delivery vehicles due, in part, to theirbiocompatibility, low immunogenicity, and low toxicity. Methods forpreparation of liposomes are known in the art and are encompassed withinthe invention, see, e.g., Eppstein et al. (1985) “Biological Activity OfLiposome-Encapsulated Murine Interferon Gamma Is Mediated By A CellMembrane Receptor,” Proc. Natl. Acad. Sci. USA, 82: 3688-3692; Hwang etal. (1980) “Hepatic Uptake And Degradation Of UnilamellarSphingomyelin/Cholesterol Liposomes: A Kinetic Study,” Proc. Natl. Acad.Sci. USA, 77: 4030-4034; U.S. Pat. Nos. 4,485,045 and 4,544,545; all ofwhich are incorporated herein by reference in their entirety.

The invention also encompasses methods of preparing liposomes with aprolonged serum half-life, i.e., enhanced circulation time, such asthose disclosed in U.S. Pat. No. 5,013,556. Preferred liposomes used inthe methods of the invention are not rapidly cleared from circulation,i.e., are not taken up into the mononuclear phagocyte system (MPS). Theinvention encompasses sterically stabilized liposomes which are preparedusing common methods known to one skilled in the art. Although notintending to be bound by a particular mechanism of action, stericallystabilized liposomes contain lipid components with bulky and highlyflexible hydrophilic moieties, which reduces the unwanted reaction ofliposomes with serum proteins, reduces oposonization with serumcomponents and reduces recognition by MPS. Sterically stabilizedliposomes are preferably prepared using polyethylene glycol. Forpreparation of liposomes and sterically stabilized liposome see, e.g.,Bendas et al. (2001) “Immunoliposomes: A Promising Approach To TargetingCancer Therapy,” BioDrugs, 15(4): 215-224; Allen et al. (1987) “LargeUnilamellar Liposomes With Low Uptake Into The ReticuloendothelialSystem,” FEBS Lett. 223: 42-46; Klibanov et al. (1990) “AmphipathicPolyethyleneglycols Effectively Prolong The Circulation Time OfLiposomes,” FEBS Lett., 268: 235-237; Blume et al. (1990) “Liposomes ForThe Sustained Drug Release In Vivo,” Biochim. Biophys. Acta., 1029:91-97; Torchilin. et al. (1996) “How Do Polymers Prolong CirculationTime Of Liposomes?,” J. Liposome Res. 6: 99-116; Litzinger et al. (1994)“Effect Of Liposome Size On The Circulation Time And IntraorganDistribution Of Amphipathic Poly(Ethylene Glycol)-Containing Liposomes,”Biochim. Biophys. Acta, 1190: 99-107; Maruyama et al. (1991) “Effect OfMolecular Weight In Amphipathic Polyethyleneglycol On Prolonging TheCirculation Time Of Large Unilamellar Liposomes,” Chem. Pharm. Bull.,39: 1620-1622; Klibanov et al. (1991) “Activity Of AmphipathicPoly(Ethylene Glycol) 5000 To Prolong The Circulation Time Of LiposomesDepends On The Liposome Size And Is Unfavorable For ImmunoliposomeBinding To Target,” Biochim Biophys Acta, 1062; 142-148; Allen et al.(1994) “The Use Of Glycolipids And Hydrophilic Polymers In AvoidingRapid Uptake Of Liposomes By The Mononuclear Phagocyte System,” Adv.Drug Deliv. Rev., 13: 285-309; all of which are incorporated herein byreference in their entirety. The invention also encompasses liposomesthat are adapted for specific organ targeting, see, e.g., U.S. Pat. No.4,544,545. Particularly useful liposomes for use in the compositions andmethods of the invention can be generated by reverse phase evaporationmethod with a lipid composition comprising phosphatidylcholine,cholesterol, and PEG derivatized phosphatidylethanolamine (PEG-PE).Liposomes are extruded through filters of defined pore size to yieldliposomes with the desired diameter. In some embodiments, a fragment ofan antibody of the invention, e.g., F(ab′), may be conjugated to theliposomes using previously described methods, see, e.g., Martin et al.(1982) “Irreversible Coupling Of Immunoglobulin Fragments To PreformedVesicles. An Improved Method For Liposome Targeting,” J. Biol. Chem.257: 286-288, which is incorporated herein by reference in its entirety.

The antibodies of the invention may also be formulated asimmunoliposomes. Immunoliposomes refer to a liposomal composition,wherein an antibody of the invention or a fragment thereof is linked,covalently or non-covalently to the liposomal surface. The chemistry oflinking an antibody to the liposomal surface is known in the art andencompassed within the invention, see, e.g., Allen et al., 1995, StealthLiposomes, Boca Rotan: CRC Press, 233-44; Hansen et al. (1995)“Attachment Of Antibodies To Sterically Stabilized Liposomes:Evaluation, Comparison And Optimization Of Coupling Procedures,”Biochim. Biophys. Acta, 1239: 133-144; which are incorporated herein byreference in their entirety. In most preferred embodiments,immunoliposomes for use in the methods and compositions of the inventionare further sterically stabilized. Preferably, the antibodies of theinvention are linked covalently or non-covalently to a hydrophobicanchor, which is stably rooted in the lipid bilayer of the liposome.Examples of hydrophobic anchors include but are not limited tophospholipids, e.g., phosoatidylethanolamine (PE), phospahtidylinositol(PI). To achieve a covalent linkage between an antibody and ahydrophobic anchor, any of the known biochemical strategies in the artmay be used, see, e.g., J. Thomas August, ed., GENE THERAPY: ADVANCES INPHARMACOLOGY, Volume 40, Academic Press, San Diego, Calif., p. 399-435,which is incorporated herein by reference in its entirety For example, afunctional group on an antibody molecule may react with an active groupon a liposome associated hydrophobic anchor, e.g., an amino group of alysine side chain on an antibody may be coupled to liposome associatedN-glutaryl-phosphatidylethanolamine activated with water-solublecarbodiimide; or a thiol group of a reduced antibody can be coupled toliposomes via thiol reactive anchors such aspyridylthiopropionyl-phosphatidylethanolamine. (See, e.g., Dietrich etal (1996) “Functional Immobilization Of A DNA-Binding Protein At AMembrane Interface Via Histidine Tag And Synthetic Chelator Lipids,”Biochemistry, 35: 1100-1105; Loughrey et al. (1987) “A Non-CovalentMethod Of Attaching Antibodies To Liposomes,” Biochim. Biophys. Acta,901: 157-160; Martin et al. (1982) “Irreversible Coupling OfImmunoglobulin Fragments To Preformed Vesicles. An Improved Method ForLiposome Targeting,” J. Biol. Chem. 257: 286-288; Martin et al. (1981)“Immunospecific Targeting Of Liposomes To Cells: A Novel And EfficientMethod For Covalent Attachment Of Fab′ Fragments Via Disulfide Bonds,”Biochemistry, 20: 4429-4438; all of which are incorporated herein byreference in their entirety.) Although not intending to be bound by aparticular mechanism of action, immunoliposomal formulations comprisingan antibody of the invention are particularly effective as therapeuticagents, since they deliver the antibody to the cytoplasm of the targetcell, i.e., the cell comprising the FcγRIIB receptor to which theantibody binds. The immunoliposomes preferably have an increasedhalf-life in blood, specifically target cells, and can be internalizedinto the cytoplasm of the target cells thereby avoiding loss of thetherapeutic agent or degradation by the endolysosomal pathway.

The invention encompasses immunoliposomes comprising an antibody of theinvention or a fragment thereof. In some embodiments, theimmunoliposomes further comprise one or more additional therapeuticagents, such as those disclosed herein.

The immunoliposomal compositions of the invention comprise one or morevesicle forming lipids, an antibody of the invention or a fragment orderivative thereof, and optionally a hydrophilic polymer. Avesicle-forming lipid is preferably a lipid with two hydrocarbon chains,such as acyl chains and a polar head group. Examples of vesicle forminglipids include phospholipids, e.g., phosphatidylcholine,phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol,sphingomyelin, and glycolipids, e.g., cerebrosides, gangliosides.Additional lipids useful in the formulations of the invention are knownto one skilled in the art and encompassed within the invention. In someembodiments, the immunoliposomal compositions further comprise ahydrophilic polymer, e.g., polyethylene glycol, and gnaglioside GM1,which increases the serum half-life of the liposome. Methods ofconjugating hydrophilic polymers to liposomes are well known in the artand encompassed within the invention. For a review of immunoliposomesand methods of preparing them, see, e.g., PCT International PublicationNo. WO 97/38731, Vingerhoeads et al. (1994) “Immunoliposomes In Vivo,”Immunomethods, 4: 259-272; Maruyama (2000) “In Vivo Targeting ByLiposomes,” Biol. Pharm. Bull. 23(7): 791-799; Abra et al. (2002) “TheNext Generation Of Liposome Delivery Systems: Recent Experience WithTumor-Targeted, Sterically-Stabilized Immunoliposomes And Active-LoadingGradients,” Journal J. Liposome Research, Res. 12(1&2): 1-3; Park (2002)“Tumor-Directed Targeting Of Liposomes,” Bioscience Reports, 22(2):267-281; Bendas et al. (2001) “Immunoliposomes: A Promising Approach ToTargeting Cancer Therapy,” BioDrugs, 14(4): 215-224, J. Thomas August,ed., GENE THERAPY: ADVANCES IN PHARMACOLOGY, Volume 40, Academic Press,San Diego, Calif., p. 399-435, all of which are incorporated herein byreference in their entireties.

Methods of administering an antibody of the invention include, but arenot limited to, parenteral administration (e.g., intradermal,intramuscular, intraperitoneal, intravenous and subcutaneous), epidural,and mucosal (e.g., intranasal and oral routes). In a specificembodiment, the antibodies of the invention are administeredintramuscularly, intravenously, or subcutaneously. The compositions maybe administered by any convenient route, for example, by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and maybe administered together with other biologically active agents.Administration can be systemic or local. In addition, pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. See, e.g., U.S.Pat. Nos. 6,019,968; 5,985, 20; 5,985,309; 5,934,272; 5,874,064;5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903, eachof which is incorporated herein by reference in its entirety.

The invention also provides that the antibodies of the invention arepackaged in a hermetically sealed container such as an ampoule orsachette indicating the quantity of antibody. In one embodiment, theantibodies of the invention are supplied as a dry sterilized lyophilizedpowder or water free concentrate in a hermetically sealed container andcan be reconstituted, e.g., with water or saline to the appropriateconcentration for administration to a subject. Preferably, theantibodies of the invention are supplied as a dry sterile lyophilizedpowder in a hermetically sealed container at a unit dosage of at least 5mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, atleast 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg. Thelyophilized antibodies of the invention should be stored at between 2and 8° C. in their original container and the antibodies should beadministered within 12 hours, preferably within 6 hours, within 5 hours,within 3 hours, or within 1 hour after being reconstituted. In analternative embodiment, antibodies of the invention are supplied inliquid form in a hermetically sealed container indicating the quantityand concentration of the antibody, fusion protein, or conjugatedmolecule. Preferably, the liquid form of the antibodies are supplied ina hermetically sealed container at least 1 mg/ml, more preferably atleast 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml,at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, at least 100mg/ml, at least 150 mg/ml, at least 200 mg/ml of the antibodies.

The amount of the composition of the invention which will be effectivein the treatment, prevention or amelioration of one or more symptomsassociated with a disorder can be determined by standard clinicaltechniques. The precise dose to be employed in the formulation will alsodepend on the route of administration, and the seriousness of thecondition, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

For antibodies encompassed by the invention, the dosage administered toa patient is typically 0.0001 mg/kg to 100 mg/kg of the patient's bodyweight. Preferably, the dosage administered to a patient is between0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kgor 0.01 to 0.10 mg/kg of the patient's body weight. Generally, humanantibodies have a longer half-life within the human body than antibodiesfrom other species due to the immune response to the foreignpolypeptides. Thus, lower dosages of human antibodies and less frequentadministration is often possible. Further, the dosage and frequency ofadministration of antibodies of the invention or fragments thereof maybe reduced by enhancing uptake and tissue penetration of the antibodiesby modifications such as, for example, lipidation.

In one embodiment, the dosage of the antibodies of the inventionadministered to a patient are 0.01 mg to 1000 mg/day, when used assingle agent therapy. In another embodiment the antibodies of theinvention are used in combination with other therapeutic compositionsand the dosage administered to a patient are lower than when saidantibodies are used as a single agent therapy.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion, by injection, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. Preferably,when administering an antibody of the invention, care must be taken touse materials to which the antibody or the fusion protein does notabsorb.

In another embodiment, the compositions can be delivered in a vesicle,in particular a liposome (See Langer (1990) “New Methods Of DrugDelivery,” Science 249:1527-1533; Treat et al., in LIPOSOMES IN THETHERAPY OF INFECTIOUS DISEASE AND CANCER, Lopez-Berestein and Fidler(eds.), Liss, New York, pp. 317-327 and 353-365 (1989)).

In yet another embodiment, the compositions can be delivered in acontrolled release or sustained release system. Any technique known toone of skill in the art can be used to produce sustained releaseformulations comprising one or more antibodies of the invention. See,e.g., U.S. Pat. No. 4,526,938; PCT publication WO 91/05548; PCTpublication WO 96/20698; Ning et al. (1996) “IntratumoralRadioimmunotheraphy Of A Human Colon Cancer Xenograft Using ASustained-Release Gel,” Radiotherapy & Oncology 39:179-189, Song et al.(1995) “Antibody Mediated Lung Targeting of Long-Circulating Emulsions,”PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek etal. (1997) “Biodegradable Polymeric Carriers for a bFGF Antibody forCardiovascular Application,” Pro. Int'l. Symp. Control. Rel. Bioact.Mater. 24:853-854; and Lam et al. (1997) “Microencapsulation ofRecombinant Humanized Monoclonal Antibody for Local Delivery,” Proc.Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which isincorporated herein by reference in its entirety. In one embodiment, apump may be used in a controlled release system (See Langer, supra;Sefton (1987) “Implantable Pumps,” CRC Crit. Ref. Biomed. Eng.14:201-240; Buchwald et al. (1980) “Long-Term, Continuous IntravenousHeparin Administration By An Implantable Infusion Pump In AmbulatoryPatients With Recurrent Venous Thrombosis,” Surgery 88:507-516; andSaudek et al. (1989) “A Preliminary Trial Of The ProgrammableImplantable Medication System For Insulin Delivery,” N. Engl. J. Med.321:574-579). In another embodiment, polymeric materials can be used toachieve controlled release of antibodies (see e.g., MEDICAL APPLICATIONSOF CONTROLLED RELEASE, Langer and Wise (eds.), CRC Pres., Boca Raton,Fla. (1974); “Controlled Drug Bioavailability,” DRUG PRODUCT DESIGN ANDPERFORMANCE, Smolen and Ball (eds.), Wiley, New York (1984); Ranger etal. (1983) “Ranger et al. (1983) “Chemical And Physical Structure OfPolymers as Carriers For Controlled Release Of Bioactive Agents: AReview,” J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126; See also Levyet al. (1985) “Inhibition Of Calcification Of Bioprosthetic Heart ValvesBy Local Controlled-Release Diphosphonate,” Science 228:190-192; Duringet al. (1989) “Controlled Release Of Dopamine From A Polymeric BrainImplant: In Vivo Characterization,” Ann. Neurol. 25:351-356; Howard etal. (1989) “Intracerebral Drug Delivery In Rats With Lesion-InducedMemory Deficits,” J. Neurosurg. 7(1):105-112); U.S. Pat. No. 5,679,377;U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No.5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154; andPCT Publication No. WO 99/20253). Examples of polymers used in sustainedrelease formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid),poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides(PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol),polyacrylamide, poly(ethylene glycol), polylactides (PLA),poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In yet anotherembodiment, a controlled release system can be placed in proximity ofthe therapeutic target (e.g., the lungs), thus requiring only a fractionof the systemic dose (see, e.g., Goodson, in MEDICAL APPLICATIONS OFCONTROLLED RELEASE, supra, vol. 2, pp. 115-138 (1984)). In anotherembodiment, polymeric compositions useful as controlled release implantsare used according to Dunn et al. (See U.S. Pat. No. 5,945,155). Thisparticular method is based upon the therapeutic effect of the in situcontrolled release of the bioactive material from the polymer system.The implantation can generally occur anywhere within the body of thepatient in need of therapeutic treatment. In another embodiment, anon-polymeric sustained delivery system is used, whereby a non-polymericimplant in the body of the subject is used as a drug delivery system.Upon implantation in the body, the organic solvent of the implant willdissipate, disperse, or leach from the composition into surroundingtissue fluid, and the non-polymeric material will gradually coagulate orprecipitate to form a solid, microporous matrix (See U.S. Pat. No.5,888,533).

Controlled release systems are discussed in the review by Langer (1990)“New Methods Of Drug Delivery,” Science 249: 1527-1533. Any techniqueknown to one of skill in the art can be used to produce sustainedrelease formulations comprising one or more therapeutic agents of theinvention. See, e.g., U.S. Pat. No. 4,526,938; International PublicationNos. WO 91/05548 and WO 96/20698; Ning et al. (1996) “IntratumoralRadioimmunotheraphy Of A Human Colon Cancer Xenograft Using ASustained-Release Gel,” Radiotherapy & Oncology 39:179-189; Song et al.(1995) “Antibody Mediated Lung Targeting Of Long-Circulating Emulsions,”PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek etal. (1997) “Biodegradable Polymeric Carriers For A bFGF Antibody ForCardiovascular Application,” Pro. Int'l. Symp. Control. Rel. Bioact.Mater. 24:853-854; and Lam et al. (1997) “Microencapsulation OfRecombinant Humanized Monoclonal Antibody For Local Delivery,” Proc.Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which isincorporated herein by reference in its entirety.

In a specific embodiment where the composition of the invention is anucleic acid encoding an antibody, the nucleic acid can be administeredin vivo to promote expression of its encoded antibody, by constructingit as part of an appropriate nucleic acid expression vector andadministering it so that it becomes intracellular, e.g., by use of aretroviral vector (See U.S. Pat. No. 4,980,286), or by direct injection,or by use of microparticle bombardment (e.g., a gene gun; Biolistic,Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (See e.g.,Joliot et al. (1991) “Antennapedia Homeobox Peptide Regulates NeuralMorphogenesis,” Proc. Natl. Acad. Sci. USA 88:1864-1868), etc.Alternatively, a nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression by homologousrecombination.

For antibodies, the therapeutically or prophylactically effective dosageadministered to a subject is typically 0.1 mg/kg to 200 mg/kg of thesubject's body weight. Preferably, the dosage administered to a subjectis between 0.1 mg/kg and 20 mg/kg of the subject's body weight, and morepreferably the dosage administered to a subject is between 1 mg/kg to 10mg/kg of the subject's body weight. The dosage and frequency ofadministration of antibodies of the invention may be reduced also byenhancing uptake and tissue penetration (e.g., into the lung) of theantibodies or fusion proteins by modifications such as, for example,lipidation.

Treatment of a subject with a therapeutically or prophylacticallyeffective amount of antibodies of the invention can include a singletreatment or, preferably, can include a series of treatments. In apreferred example, a subject is treated with antibodies of the inventionin the range of between about 0.1 to 30 mg/kg body weight, one time perweek for between about 1 to 10 weeks, preferably between 2 to 8 weeks,more preferably between about 3 to 7 weeks, and even more preferably forabout 4, 5, or 6 weeks. In other embodiments, the pharmaceuticalcompositions of the invention are administered once a day, twice a day,or three times a day. In other embodiments, the pharmaceuticalcompositions are administered once a week, twice a week, once every twoweeks, once a month, once every six weeks, once every two months, twicea year or once per year. It will also be appreciated that the effectivedosage of the antibodies used for treatment may increase or decreaseover the course of a particular treatment.

X. Pharmaceutical Composition

The compositions of the invention include bulk drug compositions usefulin the manufacture of pharmaceutical compositions (e.g., impure ornon-sterile compositions) and pharmaceutical compositions (i.e.,compositions that are suitable for administration to a subject orpatient) which can be used in the preparation of unit dosage forms. Suchcompositions comprise a prophylactically or therapeutically effectiveamount of a prophylactic and/or therapeutic agent disclosed herein or acombination of those agents and a pharmaceutically acceptable carrier.Preferably, compositions of the invention comprise a prophylactically ortherapeutically effective amount of antibodies of the invention and apharmaceutically acceptable carrier.

In one particular embodiment, the pharmaceutical composition comprisesof a therapeutically effective amount of an antibody or a fragmentthereof that binds FcγRIIB with a greater affinity than said antibody ora fragment thereof binds FcγRIIA, a cytotoxic antibody that specificallybinds a cancer antigen, and a pharmaceutically acceptable carrier. Inanother embodiment, said pharmaceutical composition further comprisesone or more anti-cancer agents.

In another particular embodiment, the pharmaceutical compositioncomprises (i) a therapeutically effective amount of an antibody orfragment thereof that specifically binds the extracellular domain ofhuman FcγRIIB and blocks the Fc binding site of human FcγRIIB; (ii) acytotoxic antibody that specifically binds a cancer antigen; and (iii) apharmaceutically acceptable carrier.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant(complete and incomplete), excipient, or vehicle with which thetherapeutic is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like.

Generally, the ingredients of compositions of the invention are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include, but are not limited tothose formed with anions such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withcaptions such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

XI. Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or fusion proteins, are administered to treat, prevent orameliorate one or more symptoms associated with a disease, disorder, orinfection, by way of gene therapy. Gene therapy refers to therapyperformed by the administration to a subject of an expressed orexpressible nucleic acid. In this embodiment of the invention, thenucleic acids produce their encoded antibody or fusion protein thatmediates a therapeutic or prophylactic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel et al.(1993) “Human Gene Therapy,” Clinical Pharmacy 12:488-505; Wu et al.(1991) “Delivery Systems For Gene Therapy,” Biotherapy 3:87-95;Tolstoshev (1993) “Gene Therapy, Concepts, Current Trials And FutureDirections,” Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan (1993)“The Basic Science Of Gene Therapy,” Science 260:926-932; and Morgan etal. (1993) “Human Gene Therapy,” Ann. Rev. Biochem. 62:191-217. Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, a composition of the invention comprises nucleicacids encoding an antibody, said nucleic acids being part of anexpression vector that expresses the antibody in a suitable host. Inparticular, such nucleic acids have promoters, preferably heterologouspromoters, operably linked to the antibody coding region, said promoterbeing inducible or constitutive, and, optionally, tissue-specific. Inanother particular embodiment, nucleic acid molecules are used in whichthe antibody coding sequences and any other desired sequences areflanked by regions that promote homologous recombination at a desiredsite in the genome, thus providing for intrachromosomal expression ofthe antibody encoding nucleic acids (Koller et al. (1989) “InactivatingThe Beta 2-Microglobulin Locus In Mouse Embryonic Stem Cells ByHomologous Recombination,” Proc. Natl. Acad. Sci. USA 86:8932-8935; andZijlstra et al. (1989) “Germ-Line Transmission Of A Disrupted B2Microglobulin Gene Produced By Homologous Recombination In EmbryonicStem Cells,” Nature 342:435-438).

In another preferred aspect, a composition of the invention comprisesnucleic acids encoding a fusion protein, said nucleic acids being a partof an expression vector that expression the fusion protein in a suitablehost. In particular, such nucleic acids have promoters, preferablyheterologous promoters, operably linked to the coding region of a fusionprotein, said promoter being inducible or constitutive, and optionally,tissue-specific. In another particular embodiment, nucleic acidmolecules are used in which the coding sequence of the fusion proteinand any other desired sequences are flanked by regions that promotehomologous recombination at a desired site in the genome, thus providingfor intrachromosomal expression of the fusion protein encoding nucleicacids.

Delivery of the nucleic acids into a subject may be either direct, inwhich case the subject is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the subject. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretroviral or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (See, e.g., Wu et al. (1987) “Receptor-Mediated In VitroGene Transformation By A Soluble DNA Carrier System,” J. Biol. Chem.262:4429-4432), which can be used to target cell types specificallyexpressing the receptors, etc. In another embodiment, nucleicacid-ligand complexes can be formed in which the ligand comprises afusogenic viral peptide to disrupt endosomes, allowing the nucleic acidto avoid lysosomal degradation. In yet another embodiment, the nucleicacid can be targeted in vivo for cell specific uptake and expression, bytargeting a specific receptor (See, e.g., PCT Publications WO 92/06180;WO 92/22635; WO92/20316; WO93/14188; WO 93/20221). Alternatively, thenucleic acid can be introduced intracellularly and incorporated withinhost cell DNA for expression, by homologous recombination (Koller et al.(1989) “Inactivating The Beta 2-Microglobulin Locus In Mouse EmbryonicStem Cells By Homologous Recombination,” Proc. Natl. Acad. Sci. USA86:8932-8935; and Zijlstra et al. (1989) “Germ-Line Transmission Of ADisrupted B2 Microglobulin Gene Produced By Homologous Recombination InEmbryonic Stem Cells,” Nature 342:435-438).

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding an antibody or a fusion protein are used. Forexample, a retroviral vector can be used (See Miller et al. (1993) “UseOf Retroviral Vectors For Gene Transfer And Expression,” Meth. Enzymol.217:581-599). These retroviral vectors contain the components necessaryfor the correct packaging of the viral genome and integration into thehost cell DNA. The nucleic acid sequences encoding the antibody or afusion protein to be used in gene therapy are cloned into one or morevectors, which facilitates delivery of the nucleotide sequence into asubject. More detail about retroviral vectors can be found in Boesen etal. (1994) “Circumvention Of Chemotherapy-Induced Myelosuppression ByTransfer Of The mdr1 Gene,” Biotherapy 6:291-302, which describes theuse of a retroviral vector to deliver the mdr 1 gene to hematopoieticstem cells in order to make the stem cells more resistant tochemotherapy. Other references illustrating the use of retroviralvectors in gene therapy include: Clowes et al. (1994) “Long-TermBiological Response Of Injured Rat Carotid Artery Seeded With SmoothMuscle Cells Expressing Retrovirally Introduced Human Genes,” J. Clin.Invest. 93:644-651; Keim et al. (1994) “Retrovirus-Mediated GeneTransduction Into Canine Peripheral Blood Repopulating Cells,” Blood83:1467-1473; Salmons et al. (1993) “Targeting Of Retroviral Vectors ForGene Therapy,” Human Gene Therapy 4:129-141; and Grossman et al. (1993)“Retroviruses: Delivery Vehicle To The Liver,” Curr. Opin. in Geneticsand Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. For a review ofadenovirus-based gene therapy see Kozarsky et al. (1993) “Gene Therapy:Adenovirus Vectors,” Current Opinion in Genetics and Development3:499-503. The use of adenovirus vectors to transfer genes to therespiratory epithelia of rhesus monkeys has been demonstrated by Bout etal. (1994) “Lung Gene Therapy: In Vivo Adenovirus-Mediated Gene TransferTo Rhesus Monkey Airway Epithelium,” Human Gene Therapy 5:3-10 Otherinstances of the use of adenoviruses in gene therapy can be found inRosenfeld et al. (1991) “Adenovirus-Mediated Transfer Of A RecombinantAlpha 1-Antitrypsin Gene To The Lung Epithelium In Vivo,” Science252:431-434; Rosenfeld et al. (1992) “In Vivo Transfer Of The HumanCystic Fibrosis Transmembrane Conductance Regulator Gene To The AirwayEpithelium,” Cell 68:143-155; Mastrangeli et al. (1993) “Diversity OfAirway Epithelial Cell Targets For In Vivo RecombinantAdenovirus-Mediated Gene Transfer,” J. Clin. Invest. 91:225-234; PCTPublication WO94/12649; and Wang et al. (1995) “A Packaging Cell LineFor Propagation Of Recombinant Adenovirus Vectors Containing Two LethalGene-Region Deletions,” Gene Therapy 2:775-783. In a preferredembodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (see, e.g., Walsh et al. (1993) “Gene Therapy For HumanHemoglobinopathies,” Proc. Soc. Exp. Biol. Med. 204:289-300).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a subject.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to, transfection, electroporation,microinjection, infection with a viral or bacteriophage vector,containing the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcellmediated gene transfer, spheroplast fusion, etc.Numerous techniques are known in the art for the introduction of foreigngenes into cells (See, e.g., Loeffler et al. (1993) “Gene Transfer IntoPrimary And Established Mammalian Cell Lines With Lipopolyamine-CoatedDNA,” Meth. Enzymol. 217:599-618, Cotten et al. (1993)“Receptor-Mediated Transport Of DNA Into Eukaryotic Cells,” Meth.Enzymol. 217:618-644) and may be used in accordance with the presentinvention, provided that the necessary developmental and physiologicalfunctions of the recipient cells are not disrupted. The technique shouldprovide for the stable transfer of the nucleic acid to the cell, so thatthe nucleic acid is expressible by the cell and preferably heritable andexpressible by its cell progeny.

The resulting recombinant cells can be delivered to a subject by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the subject.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody or a fusion protein areintroduced into the cells such that they are expressible by the cells ortheir progeny, and the recombinant cells are then administered in vivofor therapeutic effect. In a specific embodiment, stem or progenitorcells are used. Any stem and/or progenitor cells which can be isolatedand maintained in vitro can potentially be used in accordance with thisembodiment of the present invention (See e.g., PCT Publication WO94/08598; Stemple et al. (1992) “Isolation Of A Stem Cell For NeuronsAnd Glia From The Mammalian Neural Crest,” Cell 7(1):973-985; Rheinwald(1980) “Serial Cultivation Of Normal Human Epidermal Keratinocytes,”Meth. Cell Bio. 21A:229-254; and Pittelkow et al. (1986) “New TechniquesFor The In Vitro Culture Of Human Skin Keratinocytes And Perspectives OnTheir Use For Grafting Of Patients With Extensive Burns,” Mayo ClinicProc. 61:771-777).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

XII. Kits

The invention provides a pharmaceutical pack or kit comprising one ormore containers filled with antibodies of the invention. Additionally,one or more other prophylactic or therapeutic agents useful for thetreatment of a disease can also be included in the pharmaceutical packor kit. The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises one or more antibodies ofthe invention. In another embodiment, a kit further comprises one ormore other prophylactic or therapeutic agents useful for the treatmentof cancer, in one or more containers. In another embodiment, a kitfurther comprises one or more cytotoxic antibodies that bind one or morecancer antigens associated with cancer. In certain embodiments, theother prophylactic or therapeutic agent is a chemotherapeutic. In otherembodiments, the prophylactic or therapeutic agent is a biological orhormonal therapeutic.

XIII. Characterization and Demonstration of Therapeutic Utility

Several aspects of the pharmaceutical compositions or prophylactic ortherapeutic agents of the invention are preferably tested in vitro,e.g., in a cell culture system, and then in vivo, e.g., in an animalmodel organism, such as a rodent animal model system, for the desiredtherapeutic activity prior to use in humans. For example, assays whichcan be used to determine whether administration of a specificpharmaceutical composition is indicated, include cell culture assays inwhich a patient tissue sample is grown in culture, and exposed to orotherwise contacted with a pharmaceutical composition, and the effect ofsuch composition upon the tissue sample is observed, e.g., inhibition ofor decrease in growth and/or colony formation in soft agar or tubularnetwork formation in three-dimensional basement membrane orextracellular matrix preparation. The tissue sample can be obtained bybiopsy from the patient. This test allows the identification of thetherapeutically most effective prophylactic or therapeutic molecule(s)for each individual patient. Alternatively, instead of culturing cellsfrom a patient, therapeutic agents and methods may be screened usingcells of a tumor or malignant cell line. Many assays standard in the artcan be used to assess such survival and/or growth; for example, cellproliferation can be assayed by measuring ³H-thymidine incorporation, bydirect cell count, by detecting changes in transcriptional activity ofknown genes such as proto-oncogenes (e.g., fos, myc) or cell cyclemarkers; cell viability can be assessed by trypan blue staining,differentiation can be assessed visually based on changes in morphology,decreased growth and/or colony formation in soft agar or tubular networkformation in three-dimensional basement membrane or extracellular matrixpreparation, etc.

Combinations of prophylactic and/or therapeutic agents can be tested insuitable animal model systems prior to use in humans. Such animal modelsystems include, but are not limited to, rats, mice, chicken, cows,monkeys, pigs, dogs, rabbits, etc. Any animal system well-known in theart may be used. In a specific embodiment of the invention, combinationsof prophylactic and/or therapeutic agents are tested in a mouse modelsystem. Such model systems are widely used and well-known to the skilledartisan. Prophylactic and/or therapeutic agents can be administeredrepeatedly. Several aspects of the procedure may vary such as thetemporal regime of administering the prophylactic and/or therapeuticagents, and whether such agents are administered separately or as anadmixture.

Preferred animal models for use in the methods of the invention are forexample, transgenic mice expressing FcγR on mouse effector cells, e.g.,any mouse model described in U.S. Pat. No. 5,877,396 (which isincorporated herein by reference in its entirety). Transgenic mice foruse in the methods of the invention include but are not limited to micecarrying human FcγRIIIA, mice carrying human FcγRIIA, mice carryinghuman FcγRIIB and human FcγRIIIA, mice carrying human FcγRIIB and humanFcγRIIA.

Once the prophylactic and/or therapeutic agents of the invention havebeen tested in an animal model they can be tested in clinical trials toestablish their efficacy. Establishing clinical trials will be done inaccordance with common methodologies known to one skilled in the art,and the optimal dosages and routes of administration as well as toxicityprofiles of the compositions of the invention can be established usingroutine experimentation.

Toxicity and efficacy of the prophylactic and/or therapeutic protocolsof the instant invention can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Prophylacticand/or therapeutic agents that exhibit large therapeutic indices arepreferred. While prophylactic and/or therapeutic agents that exhibittoxic side effects may be used, care should be taken to design adelivery system that targets such agents to the site of affected tissuein order to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the prophylactic and/ortherapeutic agents for use in humans. The dosage of such agents liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any agent used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

The anti-cancer activity of the therapies used in accordance with thepresent invention also can be determined by using various experimentalanimal models for the study of cancer such as the SCID mouse model ortransgenic mice or nude mice with human xenografts, animal models, suchas hamsters, rabbits, etc. known in the art and described in “Relevanceof Tumor Models for Anticancer Drug Development” (1999) Fiebig andBurger, eds., CONTRIBUTIONS TO ONCOLOGY; THE NUDE MOUSE IN ONCOLOGYRESEARCH, 1991, Boven and Winograd, eds.; and ANTICANCER DRUGDEVELOPMENT GUIDE, (1997) Teicher, ed., herein incorporated by referencein their entireties.

The protocols and compositions of the invention are preferably tested invitro, and then in vivo, for the desired therapeutic or prophylacticactivity, prior to use in humans. Therapeutic agents and methods may bescreened using cells of a tumor or malignant cell line. Many assaysstandard in the art can be used to assess such survival and/or growth;for example, cell proliferation can be assayed by measuring ³H-thymidineincorporation, by direct cell count, by detecting changes intranscriptional activity of known genes such as proto-oncogenes (e.g.,fos, myc) or cell cycle markers; cell viability can be assessed bytrypan blue staining, differentiation can be assessed visually based onchanges in morphology, decreased growth and/or colony formation in softagar or tubular network formation in three-dimensional basement membraneor extracellular matrix preparation, etc.

Compounds for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to inrats, mice, chicken, cows, monkeys, rabbits, hamsters, etc., forexample, the animal models described above. The compounds can then beused in the appropriate clinical trials.

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of thecombinatorial therapies disclosed herein for treatment or prevention ofcancer.

XIV. Diagnostic Methods

Labeled antibodies of the invention can be used for diagnostic purposesto detect, diagnose, or monitor diseases, disorders or infections. Theinvention provides for the detection or diagnosis of a disease, disorderor infection comprising: (A) assaying the expression of FcγRIIB in cellsor a tissue sample of a subject using one or more antibodies thatimmunospecifically bind to FcγRIIB; and (B) comparing the level of theantigen with a control level, e.g., levels in normal tissue samples,whereby an increase in the assayed level of antigen compared to thecontrol level of the antigen is indicative of the disease, disorder orinfection.

Antibodies of the invention can be used to assay FcγRIIB levels in abiological sample using classical immunohistological methods asdescribed herein or as known to those of skill in the art (see e.g.,Jalkanen et al. (1985) “Heparan Sulfate Proteoglycans From Mouse MammaryEpithelial Cells: Localization On The Cell Surface With A MonoclonalAntibody,” J. Cell. Biol. 101:976-984; Jalkanen et al. (1987) “CellSurface Proteoglycan Of Mouse Mammary Epithelial Cells Is Shed ByCleavage Of Its Matrix-Binding Ectodomain From Its Membrane-AssociatedDomain,” J. Cell. Biol. 105:3087-3096). Other antibody-based methodsuseful for detecting protein gene expression include immunoassays, suchas the enzyme linked immunosorbent assay (ELISA) and theradioimmunoassay (RIA). Suitable antibody assay labels are known in theart and include enzyme labels, such as, alkaline phosphatase, glucoseoxidase; radioisotopes, such as iodine (¹²⁵I, ¹³¹I), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (¹²¹In), and technetium (⁹⁹M Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine.

One aspect of the invention is the detection and diagnosis of a disease,disorder, or infection in a human. In one embodiment, diagnosiscomprises: (A) administering (for example, parenterally, subcutaneously,or intraperitoneally) to a subject an effective amount of a labeledantibody that immunospecifically binds to FcγRIIB; (B) waiting for atime interval following the administration for permitting the labeledantibody to preferentially concentrate at sites in the subject whereFcγRIIB is expressed (and for unbound labeled molecule to be cleared tobackground level); (C) determining background level; and (D) detectingthe labeled antibody in the subject, such that detection of labeledantibody above the background level indicates that the subject has thedisease, disorder, or infection. In accordance with this embodiment, theantibody is labeled with an imaging moiety which is detectable using animaging system known to one of skill in the art. Background level can bedetermined by various methods including, comparing the amount of labeledmolecule detected to a standard value previously determined for aparticular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of ^(99m)Tc. The labeled antibodywill then preferentially accumulate at the location of cells whichcontain the specific protein. In vivo tumor imaging is described in S.W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodiesand Their Fragments.” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6-48 hours or 6-24 hours or 6-12 hours. In anotherembodiment, the time interval following administration is 5 to 20 daysor 5-10 days.

In one embodiment, monitoring of a disease, disorder or infection iscarried out by repeating the method for diagnosing the disease, disorderor infection, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the subject usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patient using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Example 1 Preparation Of Monoclonal Antibodies

A mouse monoclonal antibody was produced from clones 3H7 or 2B6 withATCC accession numbers PTA-4591 and PTA-4592, respectively. A mousemonoclonal antibody that specifically binds FcγRIIB with greateraffinity than said monoclonal antibody binds FcγRIIA, was generated.Transgenic FcγRIIA mice (generated in Dr. Ravetch Laboratory,Rockefeller University) were immunized with FcγRIIB purified fromsupernatant of 293 cells that had been transfected with cDNA encodingthe extracellular domain of the human FcγRIIB receptor, residues 1-180.Hybridoma cell lines from spleen cells of these mice were produced andscreened for antibodies that specifically bind FcγRIIB with greateraffinity than the antibodies bind FcγRIIA.

Antibody Screening and Characterization

Materials And Methods

Supernatants from hybridoma cultures are screened for immunoreactivityagainst FcγRIIA or FcγRIIB using ELISA assays. In each case, the plateis coated with 100 ng/well of FcγRIIA or FcγRIIB. The binding of theantibody to the specific receptor is detected with goat anti-mouse HRPconjugated antibody by monitoring the absorbance at 650 nm.

In the blocking ELISA experiment, the ability of the antibody from thehybridoma supernatant to block binding of aggregated IgG to FcγRIIB ismonitored. The plate is blocked with the appropriate “blocking agent”,washed three times (200 μl/well) with wash buffer (PBS plus 0.1% Tween).The plate is pre-incubated with hybridoma supernatant for 1 hour at 37°C. Subsequent to blocking, a fixed amount of aggregated biotinylatedhuman IgG (1 μg/well) is added to the wells to allow the aggregate tobind to the FcγRIIB receptor. This reaction is carried out for two hoursat 37° C. Detection is then monitored, after additional washing, withstreptavidin horseradish peroxidase conjugate, which detects the boundaggregated IgG. The absorbance at 650 nm is proportional to the boundaggregated IgG.

In a β-hexoaminidase release assay the ability of an antibody from thehybridoma supernatant to inhibit Fcε-induced release of β-hexoaminidaseis monitored. RBL-2H3 cells are transfected with human FcγRIIB; cellsare stimulated with various concentration of goat anti-mouse F(ab)₂fragment ranging from 0.03 μg/mL to 30 μg/mL; sensitized with eithermouse IgE alone (at 0.01 μg/mL) or with an anti-FcγRIIB antibody. After1 hour incubation at 370 temperature, the cells are spun down; thesupernatant is collected; and the cells are lysed. The β-hexoaminidaseactivity released in the supernatant is determined in a colorometricassay using p-nitrophenyl N-acetyl-βD-glucoasminide. The releaseβ-hexoaminidase activity is expressed as a percentage of the releasedactivity relative to the total activity.

FACS Analysis:

CHO cells, expressing FcγRIIB are stained with various antibodies andanalyzed by FACS. In one series of experiment, the cells are directlylabeled to determine if the monoclonal antibodies recognize thereceptor.

In the blocking FACS experiment, the ability of the antibody from thehybridoma supernatant to block the binding of aggregated IgG to FcγRIIBis monitored. About 1 million cells (CHO cells expressing FcγRIIB) foreach sample are incubated on ice for 30 minutes with 2 μg of the isotypecontrol (mouse IgG1) or with the 2B6 or 3H7 antibody. Cells are washedonce with PBS+1% BSA and incubated with 1 μg of aggregated biotinylatedhuman IgG for 30 minutes on ice. Cells are washed and the secondaryantibodies are added, goat anti-mouse-FITC to detect the bound antibodyand Streptavidin-PE conjugated to detect the bound aggregatedbiotinylated human IgG and incubated on ice for 30 minutes. Cells arewashed and analyzed by FACS.

B Lymphocytes are stained to detect the presence of FcγRIIB and CD20.200 μl of “buffy coat” for each sample is incubated on ice with 2 μg ofisotype control or the monoclonal antibodies, 2B6 or 3H7. Cells arewashed once with PBS+1% BSA and incubated with 1 μl of goat antimouse-PE antibody for 30 minutes on ice. Cells are washed once andCD20-FITC antibody (2 μg) is added to the samples and incubated on icefor 30 minutes. All samples are washed with PBS+1% BSA once and thecells are analyzed by FACS.

Human PBMCs were stained with 2B6, 3H7, and IV.3 antibodies, followed bya goat anti-mouse-Cyanine (Cy5) conjugated antibody (two color stainingusing anti-CD20-FITC conjugated for B lymphocytes, anti-CD14-PEconjugated for monocytes, anti-CD56-PE conjugated for NK cells andanti-CD16-PE conjugated for granulocytes.

ADCC Assay:

4-5×10⁶ target cells expressing Her2/neu antigen (IGROV-1 or cells) arelabeled with bis(acetoxymethyl)2,2′:6′,2″-terpyridine-t-6″-dicarboxylate (DELFIA BATDA Reagent, PerkinElmer/Wallac). BATDA reagent is added to the cells and the mixture isincubated at 37° C. preferably under 5% CO₂, for at least 30 minutes.The cells are then washed with a physiological buffer, e.g., PBS with0.125 mM sulfinpyrazole, and media containing 0.125 mM sulfinpyrazole.The labeled target cells are added to effector cells, e.g., PBMC, toproduce effector:target ratios of approximately 50:1, 75:1, or 100:1.PBMC is isolated by layering whole blood onto Ficoll-Hypaque (Sigma) andspinning at room temperature for 30 mins at 500 g. The leukocyte layeris harvested as effectors for Europium-based ADCC assays. Frozen orfreshly isolated elutriated monocytes (Advanced Biotechnologies, MD) isused as effectors with the tumor target cell lines at varying effectorto target ratio of 100:1 to 10:1 and the concentration of the antibodiesis titrated from 1-15 μg/ml. Monocytes obtained as frozen stocksstimulated with cytokines is used as effector cells in ADCC assays. Iffrozen monocytes perform optimally they will be routinely used otherwisefresh cells will be used. MDM will be prepared by treatment withcytokines GM-CSF or M-CSF that are known to enhance the viability anddifferentiation of monocytes in culture. MDM will be stimulated withcytokines and the expression of the various FcγRs (I, IIA, IIB, and111A) determined by FACS analysis.

The effector and target cells are incubated for at least two hours, andup to 16 hours, at 37° C., under 5% CO₂ in the presence of an anti-tumorantibody, specific for an antigen expressed on the target cells,Her2/neu, and in the presence or absence of an anti-FcγRIIB antibody. Achimeric 4D5 antibody that has been engineered to contain the N297Amutation which is used as a negative control since this antibody bindsthe tumor target cells via its variable region. Loss of glycosylation atthis site abolishes binding of the Fc region of the antibody to FcγR.Commercially available human IgG1/k serves as an isotype control for theanti-FcγRIIB antibody. Cell supernatants are harvested and added to anacidic europium solution (e.g., DELFIA Europium Solution, PerkinElmer/Wallac). The fluorescence of the Europium-TDA chelates formed isquantitated in a time-resolved fluorometer (e.g., Victor 1420, PerkinElmer/Wallac). Maximal release (MR) and spontaneous release (SR) aredetermined by incubation of target cells with 1% TX-100 and media alone,respectively. Antibody independent cellular cytotoxicity (AICC) ismeasured by incubation of target and effector cells in the absence ofantibody. Each assay is preferably performed in triplicate. The meanpercentage specific lysis is calculated as: Experimental release(ADCC)−AICC)/(MR−SR)×100.

Example 2 Characterization of the Monoclonal Antibody Produced from the3H7 Clone

The direct binding of different batches of hybridoma cultures to FcγRIIAand FcγRIIB were compared using an ELISA assay (FIG. 1A). Supernatantsnumbered 1, 4, 7, 9, and 3 were tested for specific binding and theirbinding was compared to a commercially available antibody, FL18.26. Asshown in FIG. 1A (left panel), supernatant from clone 7 has the maximalbinding to FcγRIIB, which is about four times higher under saturatingconditions than the binding of the commercially available antibody toFcγRIIB. However, the supernatant from clone 7 has hardly any affinityfor FcγRIIA, as seen in the right panel, whereas the commerciallyavailable antibody binds FcγRIIA at least 4 times better.

Direct binding of the antibody produced from the 3H7 clone to FcγRIIAand FcγRIIB. The binding of crude 3H7 supernatant and purified 3H7supernatant was measured (FIG. 1B). In each case, the supernatant wassupplied at a concentration of 70 μg/ml and diluted up to 6-fold. Asshown in FIG. 1B, upon saturating conditions, the 3H7 supernatant bindsFcγRIIB four times better than it binds FcγRIIA. Upon purification withan protein G column, the absolute binding of the 3H7 supernatant to eachimmunogen improves.

Blocking of aggregated human IgG binding to FcγRIIB by the antibodyproduced from the 3H7 clone. If the antibody present in the hybridomasupernatant binds FcγRIIB at the IgG binding site and blocks IgGbinding, then the aggregated IgG cannot bind the receptor and hence noabsorbance at 650 can be detected. The antibody in effect is a “blockingagent” that blocks the IgG binding site on FcγRIIB. As a control, theELISA was carried out with no blocking, with a control supernatant, andwith supernatant from the 3H7 clone. As shown in FIG. 2, the 3H7supernatant completely blocks IgG binding, since aggregated IgG cannotbind the receptor as evident from the lack of absorbance at 650 nm. Thecontrol supernatant however fails to block IgG binding; aggregated IgGbinds the receptor as evident by the reading at 650 nm. The controlsupernatant behaves similarly to the condition where no blocking wasdone.

Comparison of the direct binding of the antibody produced from the 3H7clone to bacterial and mammalian FcγRIIB. As shown in FIG. 3, thesupernatant from the 3H7 clone, binds comparably to mammalian andbacterial FcγRIIB. Upon saturating conditions, the 3H7 supernatant bindsbacterial and mammalian FcγRIIB about three times better than it bindsFcγRIIA. The monoclonal antibody from the 3H7 clone is thus able tospecifically bind to mammalian FcγRIIB which has beenpost-transnationally modified (e.g., glycosylation).

Direct binding of the antibody produced from the 3H7 clone to FcγRIIA,FcγRIIB, and FcγRIIIA. The direct binding of supernatant from thehybridoma cultures from the 3H7 cell line to FcγRIIA, FcγRIIIA andFcγRIIB were compared using an ELISA assay (FIG. 4). The antibodyproduced from clone 3H7 has no affinity for FcγRIIIA, and binds FcγRIIBwith about 4 times greater affinity than it binds FcγRIIA.

Example 3 Characterization of the Monoclonal Antibody Produced from the2B6 Clone

Comparison of direct binding of the antibody produced from clone 2B6compared to other three commercially available monoclonal antibodiesagainst FcγRII. The binding of the antibody produced from clone 2B6 toFcγRIIA and FcγRIIB is compared to that of three other commerciallyavailable antibodies, AT10, FL18.26, and IV.3, against FcγRII in anELISA assay. As seen in FIG. 5A, the antibody produced from clone 2B6binds FcγRIIB up to 4.5 times better than the other commerciallyavailable antibodies. Additionally, the antibody produced from clone 2B6has minimal affinity for FcγRIIA, whereas the other three commerciallyavailable antibodies bind FcγRIIA in a saturatable manner and twice asmuch as the antibody from clone 2B6 binds FcγRIIA (FIG. 5B).

Blocking of aggregated human IgG to FcγRIIB by the antibody producedfrom clone 2B6. The ability of the antibody produced from clone 2B6 toblock binding of the aggregated IgG to FcγRIIB was investigated by ablocking ELISA assay and compared to that of the antibody produced byclone 3H7. As shown in FIG. 6A, the control supernatant does not bindFcγRIIB on the IgG binding site and the aggregated IgG can bind thereceptor and hence absorbance at 650 nm is maximal. Clone 3H7, however,blocks the IgG binding up to 75%. Clone 2B6 completely blocks thebinding of the IgG binding site and does not allow the aggregated IgG tobind the receptor, and even at very high dilutions no absorbance isdetected at 650 nm. FIG. 6B represents the data in a bar diagram.

Competition of 2B6 antibody and aggregated IgG in binding FcγRIIB usingdouble-staining FACS assays. A double staining FACS assay was used tocharacterize the antibody produced from clone 2B6 in CHO cells that hadbeen transfected with full-length mammalian FcγRIIB.

As shown in FIG. 7C, the antibody produced from clone 2B6 effectivelyblocks the binding of aggregated IgG to the FcγRIIB receptor in CHOcells since no staining is observed for biotinylated aggregated IgGafter the cells were pre-incubated with the monoclonal antibody. Thecells are only stained in the lower right panel, indicating that most ofthe cells were bound to the monoclonal antibody from the 2B6 clone. Inthe control experiments, using IgG1 as the isotype control, FIG. 7A,when the cells are stained with the isotype labeled IgG, no staining isobserved since the monomeric IgG does not bind FcγRIIB with anydetectable affinity, whereas in FIG. 7B, about 60% of the cells arestained with aggregated IgG, which is capable of binding FcγRIIB.

Monoclonal anti-FcγRIIB antibodies and CD20 co-stain Human BLymphocytes. A double staining FACS assay was used to characterize theantibody produced from clones 2B6 and 3H7 in human B lymphocytes. Cellswere stained with anti-CD20 antibody which was FITC conjugated, toselect the B-lymphocyte population, as well as the antibodies producedfrom clone 3H7 and 2B6, labeled with goat anti-mouse peroxidase. Thehorizontal axis represents the intensity of the anti-CD20 antibodyfluorescence and the vertical axis represents the intensity of themonoclonal antibody fluorescence. As shown in FIGS. 8B and 8C, cells aredouble stained with the anti-CD20 antibody as well as the antibodiesproduced from clones 2B6 and 3H7, however, the antibody produced fromclone 2B6 shows more intense staining than that produced from clone 3H7.FIG. 8A shows the staining of the isotype control, mouse IgG1.

Staining of CHO cells expressing FcγRIIB. CHO cells, stably expressingFcγRIIB were stained with IgG1 isotype control (FIG. 9) or withsupernatant from the 3H7 hybridoma (FIG. 9). Goat anti-mouse peroxidaseconjugated antibody was used as a secondary antibody. The cells werethen analyzed by FACS; cells that are stained with the supernatant fromthe 3H7 hybridoma show a strong fluorescence signal and a peak shift tothe right; indicating the detection of FcγRIIB in the CHO cells by thesupernatant produced from the 3H7 hybridoma. Cells stained with thesupernatant from the 2B6 hybridoma, also show a significantfluorescence, as compared to cells stained with IgG1, and a peak shiftto the right, indicating the detection of FcγRIIB in the CHO cells bythe supernatant produced from the 2B6 hybridoma.

CHO cells expressing hyFcγRIIB were incubated with the anti CD32Bantibodies, 2B6 or 3H7. Cells were washed and 9 μg/ml of aggregatedhuman IgG were added to the cells on ice. The human aggregated IgG weredetected with goat anti human-IgG GITC conjugated. Samples were analyzedby FACS cells labeled with 2B6 or 3H7 showed a significant fluorescencepeak in the presence of aggregated human IgG (FIG. 10). 2BG antibodycompletely blocks binding of aggregated IgG as evidenced by thefluorescent peak shift to the left. Whereas the 3H7 antibody partiallyblocks binding of aggregated IgG as shown by the intermediatefluorescent peak. The other antibodies, 1D5, 1F2, 2E1, 2H9, and 2D11 donot block binding of aggregated IgG (FIG. 10). The amount of eachantibody bound to the receptor on the cells was also detected (inset) ona separate set of samples using a goat anti-mouse PE conjugatedantibody.

FACS profiles using 2B6, 3H7, and IV.3 antibodies on human peripheralblood leukocyte. The FACS profile of the anti-FcγRIIB antibodies andIV.3 antibody shows their ability to discriminate between the two FcγRIIisoforms, IIB and IIA expressed on the human hematopoietic cells. IV.3,one of the first antibodies (commercially available) used to defineFcγRII, shows preferential binding to FcγRIIA.

There are characteristic and functionally significant differences inisoform expression between major human hematopoietic cell types. Human Blymphocytes express exclusively the huFcγRIIB isoform while humanmonocytes express predominantly the huFcγRIIA isoform. Granulocytes arestrongly positive for FcγRIIA and limited evidence suggest that FcγRIIBis marginally expressed in this population (Pricop et al, (2000)“Differential Modulation Of Stimulatory And Inhibitory Fc GammaReceptors On Human Monocytes By Th1 And Th2 Cytokines,” J. Immunol.166:531-537). To further characterize the reactivity of the anti-FcγRIIBantibodies, huPBL were stained with the anti-FcγRIIB antibodies 2B6 and3H7 and with IV.3, which preferentially (but not exclusively) recognizesthe FcγRIIA isoform of the receptor, leukocytes populations wereselected based on FSC vs. SSC gating (FIGS. 11A-11P) and identified withspecific markers: CD20 (B cells) (FIGS. 11B, 11D and 11F), CD56 (NKcells) (FIGS. 11I and 11J) or CD16 (lymphocyte gate), CD14 (monocytes)(FIGS. 11K, 11M and 11O) and CD16 (granulocytes, granulocyte gate)(FIGS. 11C, 11E, 11G, 11L, 11N and 11P). CD20-positive cells (B cells)were uniformly stained with 2B6, 3H7. IV.3 also stained the majority ofCD20-positive cells. No staining was observed for CD16/CD56-positive NKcells, while only a fraction of CD14-(monocytes) and CD16-(granulocytes)positive cells were stained with 2B6, 3H7. In contrast, IV.3 stronglystained the vast majority of CD-14-positive monocytes and the totalityof CD16-positive granulocytes (FIGS. 11J and 11O-P). This differentialpattern of reactivity between 2B6 and 3H7 on the one side and IV.3 onthe other indicates that the new monoclonal antibodies react stronglywith FcγRIIB, but not with FCγRIIA, while IV.3 cannot discriminatebetween FcγRIIA and FcγRIIB isoforms in vivo.

Inhibition Of β-Hexosaminidase Release By 2B6. To examine the potentialrole of an anti-CD32B antibody in modulating immediate-typehypersensitivity reactions, the effect of inducing a co-aggregation ofactivating (FcεRI) and inhibitory receptors (FcγRIIB) was investigated.The rat basophilic leukemia cell line, RBL-2H3, was chosen as a modelsystem due its extensive use in the art as an allergy model designed tostudy the underlying mechanism of IgE-mediated mast cell activation (Ottet al. (2002) “Downstream of Kinase, p62dok, Is a Mediator of FcRIIBInhibition of FcRI Signaling,” J. Immunol. 168:4430-9). FIG. 12B:Transfected RBL cells expressing FcγRIIB were suspended in fresh mediacontaining 0.01 μg/ml of murine anti-DNP IgE and plated in 96 wellplates at a concentration of 2×10⁴ cells/well. After over-nightincubation at 37° C. in the presence of CO₂, cells were washed twicewith pre-warmed release buffer (10 mM HEPES, 137 mM NaCl, 2.7 mM KCl,0.4 mM sodium phosphate monobasic, 5.6 mM glucose, 1.8 mM calciumchloride, 1.3 mM magnesium sulfate and 0.04% BSA, pH 7.4) and treated at37° C. with serial dilutions of BSA-DNP-FITC complexed with chimeric4-4-20 antibody or BSA-DNP-FITC complexed with chimeric D265A 4-4-20antibody in 100 μl buffer/well in the presence of 2B6 antibody, 1F2antibody or murine IgG1 isotype control. Alternatively cells werechallenged with F(ab′)₂ fragments of a polyclonal goat anti-mouse IgG toaggregate FcεRI (Genzyme). Crosslinking of the FcεRs occurs because thepolyclonal antibody recognizes the light chain of the murine IgEantibody bound to FcεRI. This experiment is schematically shown in FIG.12A.

The reaction was stopped after 30 minutes by placing the cells on ice.50 μl of supernatant from each well was removed and the cells wereosmotically lysed. Cell lysates were incubated withp-Nitrophenyl-N-Acetyl-beta-D-glucosaminide (5 mM) for 90 minutes, thereaction was stopped with glycine (0.1M, pH 10.4) and the absorbance at405 nm was measured after three minutes. The percentage ofβ-hexosaminidase released was calculated as total media OD/totalsupernatant OD/total supernatant+total cell lysate OD.

RESULTS. To test the ability of ch2B6 to limit the inflammatory orallergic responses triggered by the activating receptor, F(ab′)₂fragments were used to coaggregate activating receptors or combinationsof inhibitory and activating receptors as described above. When cellswere sensitized only with IgE, the F(ab′)₂ fragments of polyclonal goatanti-mouse IgG recognized the light chain of the murine IgE bound toFcεRI, aggregated these activating receptors, and β-hexosaminidaserelease, a marker for degranulation (Aketani et al. (2001) “CorrelationBetween Cytosolic Calcium Concentration And Degranulation InRBL-2H3Cells In The Presence Of Various Concentrations OfAntigen-Specific IgEs,” Immunol. Lett. 75: 185-189), increased withincreasing IgE (FIG. 12B). In contrast, when cells were sensitized withIgE after incubation with 2B6 or 1F2, the F(ab′)₂ fragment, in effect,co-cross-linked the rat FcεRI with CD32B and resulted in a significantdecrease in β-hexosaminidase release when compared to sensitized cellspreincubated with an irrelevant murine IgG₁ isotype control matchedantibody. No degranulation over background levels was detected in cellstreated with the anti-CD32B antibodies alone (data not shown).Therefore, the human inhibitory receptor, CD32B, can induce a negativesignal in rat basophilic cells, validating these transfectants as amodel for the study of anti-human CD32B antibodies.

To test whether anti-CD32B antibodies may also be able to improve suchreactions, the co-engagement of the inhibitory receptor with anactivating receptor was prevented by a blockade of CD32B. Co-engagementof these receptors is thought to physiologically occur when antigenssimultaneously interact with surface-bound IgE through antigenicepitopes and with CD32B through Fc determinants of antigen-specific IgGcomplexed with the antigen itself (FIG. 13A). To mimic this situation,the RBL-2H3 model was manipulated to obtain co-engagement of FcεRI andCD32B by developing an antigen surrogate that could be complexed withIgE, IgG, or both. HuCD32B⁺ RBL-2H3 cells were sensitized with a murineIgE anti-DNP monoclonal antibody. The challenge antigen, BSA-DNP, wasfurther conjugated to FITC to provide additional epitopes recognized bya chimeric version of 4-4-20, a murine anti-fluorescein antibody whoseFc portion had been substituted with human IgG₁ Fc to allow for optimalbinding to human CD32B. A chimeric version of 4-4-20 with a human IgG₁Fc bearing a mutation in position 265 (asparagine to alanine) was alsogenerated. This chimeric D265A 4-4-20 antibody lacks the ability to bindFcγR's, including CD32B. BSA-DNP-FITC induced a dose-dependent releaseof β-hexosaminidase from IgE-sensitized RBL-2H3 cells (FIG. 13C).

The same extent of degranulation was observed when the challenge antigenwas BSA-DNP-FITC complexed with chimeric D265A 4-4-20, showing thatBSA-DNP-FITC-chimeric D265A 4-4-20, as expected, was unable to recruitCD32B to the activating receptor. In the presence of BSA-DNP-FITCcomplexed with chimeric 4-4-20, a substantial reduction inβ-hexosaminidase release was observed (FIG. 13B). Thus, the polyvalentantigen is capable of aggregating FcεRI with ensuing degranulation,while the surrogate antigen complexed with IgG co-aggregates CD32Bresulting in diminished degranulation. To block CD32B while minimizingthe chances of simultaneously engaging the FcγR, F(ab)₂ fragments of 2B6where prepared and cells pre-incubated with 2B6 F(ab)₂, prior toactivation with the immunocomplexed antigen. Under these conditions, thepercentage of β-hexosaminidase release was restored to the maximumlevels observed in cells treated with the polyvalent antigen alone (FIG.13C). At higher concentrations of immunocomplexed antigen a diminisheddegranulation was still observed, presumably due to competition betweench4-4-20 and 2B6 F(ab)₂ for the Fc binding site of CD32B. These datashow that 2B6 is capable of functionally blocking the Fc binding site ofCD32B, preventing the co-ligation of activating and inhibitory receptorsby an IgG-complexed antigen. The proposed mode of action may have use inthe regulation of immunecomplex-mediated cell activation.

Example 4 Her2/neu Expression in Ovarian and Breast Carcinoma Cell Lines

In order to determine whether IGROV-1, OVCAR-8, and SKBR-3 cells expressthe Her2/neu antigen, cells were stained with either purified 4D5 orch4D5 antibody on ice; the unbound antibody was washed out with PBS/BSAbuffer containing sodium azide, and the binding of 4D5 or ch4D5 wasdetected by goat anti-mouse or goat anti-human antibody conjugated to PE(Jackson Laboratories), respectively. An irrelevant IgG1 antibody(Becton Dickinson) served as a control for non-specific binding. Asshown in FIGS. 14A-14C, the ovarian tumor cell lines express lessHer2/neu antigens than the breast carcinoma cell line and evaluatingthese cell lines in parallel will determine the stringency of tumorclearance by an anti-FcγRIIB antibody of the invention.

Human monocytes are the effector population involved in ADCC thatexpress both activating and inhibitory receptors. The expression ofFcγRs was tested by FACS analysis using several lots of frozen monocytesas these cells will be adoptively transferred as effectors toinvestigate the role of ch2B6 in tumor clearance. Commercially obtainedfrozen elutriated monocytes were thawed in basal medium containing 10%human AB serum and in basal medium with human serum and 25-50 ng/mlGM-CSF. Cells were either stained directly or allowed to mature tomacrophages for 7-8 days (MDM), lifted off the plastic, and then stainedwith IV.3-FITC (anti-hu FcγRIIA), 32.2-FITC (anti-FcγRI), CD16-PE(Pharmingen) or 3G8 (anti-FcγRII)-goat anti-mouse-PE, 3H7(anti-FcγRIIB), and CD14 marker for monocytes (Pharmingen), along withrelevant isotype controls. A representative FACS profile of MDM from twodonors, depicting FcγR expression on freshly thawed monocytes andcultured monocytes, is shown in FIGS. 15A-15C.

These results indicate that FcγRIIB is modestly expressed in monocytes(5-30% depending on the donor). However this expression increases asthey mature into macrophages. Preliminary data show thattumor-infiltrating macrophages in human tumor specimens are positivelystained for FcγRIIB (data not shown). The pattern of FcγRs and theability to morphologically differentiate into macrophages was found tobe reproducible in several lots of frozen monocytes. These data indicatethat this source of cells is adequate for adoptive transfer experiments.

Ch4D5 mediates effective ADCC with ovarian and breast cancer cells linesusing PBMC. The ADCC activity of anti-Her2/neu antibody was tested in aeuropium based assay. The ovarian cell line, IGROV-1, and the breastcancer cell line, SKBR-3, were used as labeled targets in a 4 hour assaywith human PBL as effector cells. FIGS. 16A and 16B indicate that ch4D5is functionally active in mediating lysis of targets expressingHer2/neu. The effect of an antibody of the invention on the ADCCactivity of the anti-Her2/neu antibody is subsequently measured.

Example 5 In Vitro ADCC Assay

A chimeric anti-CD32B antibody (ch2B6) and its aglycosylated form(ch2B6Agly) were tested for the ability to mediate in vitro antibodydependent cell-mediated cytotoxicity (ADCC) against CD32B-expressing,B-cell lymphoma lines, Daudi and Raji.

The protocol for assessment of antibody dependent cellular cytotoxicity(ADCC) is similar to that previously described in (Ding et al. (1998)“Two Human T Cell Receptors Bind In A Similar Diagonal Mode To TheHLA-A2/Tax Peptide Complex Using Different TCR Amino Acids,” Immunity8:403-411) and described herein. Briefly, target cells from the CD32Bexpressing B-cell lymphoma lines, Daudi and Raji, were labeled with theeuropium chelate bis(acetoxymethyl)2,2′:6′,2″-terpyridine-6,6″-dicarboxylate (DELFIA BATDA Reagent, PerkinElmer/Wallac). The labeled target cells were then opsonized (coated)with either chimeric anti-CD32B (ch2B6) or aglycosylated chimericanti-CD32B (ch2B6Agly) antibodies at the indicated concentrations asshown in FIGS. 18 and 19. Peripheral blood mononuclear cells (PBMC),isolated by Ficoll-Paque (Amersham Pharmacia) gradient centrifugation,were used as effector cells (Effector to Target ratio of 75 to 1).Following a 3.5 hour incubation at 37° C., 5% CO₂, cell supernatantswere harvested and added to an acidic europium solution (DELFIA EuropiumSolution, Perkin Elmer/Wallac). The fluorescence of the Europium-TDAchelates formed was quantitated in a time-resolved fluorometer (Victor²1420, Perkin Elmer/Wallac). Maximal release (MR) and spontaneous release(SR) were determined by incubation of target cells with 2% Triton X-100and media alone, respectively. Antibody independent cellularcytotoxicity (AICC) was measured by incubation of target and effectorcells in the absence of antibody. Each assay is performed in triplicate.The mean percentage specific lysis is calculated as:(ADCC−AICC)/(MR−SR)×100.

As shown in FIGS. 18 and 19, chimeric anti-CD32B antibody ch2B6 mediatesADCC in vitro against CD32B-expressing, B-cell lymphoma lines, Daudi andRaji, at concentrations greater than approximately 10 ng/ml. Thisactivity is likely to be Fc-dependent since the aglycoslyated version ofthis antibody, ch2B6Agly, which is unable to interact with theFc-receptors has reduced activity in this assay.

Example 6 In Vivo ADCC Assay

Six to eight week old female Balb/c nude mice (Jackson Laboratories, BarHarbor, Me.; Taconic) is utilized for establishing the xenograft ovarianand breast carcinoma models. Mice are maintained at BIOCON, Inc.Rockville, Md. (see attached protocol). Mice are housed in BiosafetyLevel-2 facilities for the xenograft model using the ascites-derivedovarian cells and pleural effusion-derived breast cancer cells assources of tumors. Mice are placed in groups of 4 for these experimentsand monitored three times weekly. The weight of the mice and survivaltime are recorded and criteria for growing tumors is abdominaldistention and palpable tumors. Mice showing signs of visible discomfortor that reach 5 grams in tumor weight are euthanized with carbon dioxideand autopsied. The antibody-treated animals are placed under observationfor an additional two months after the control group.

Establishment of the xenograft tumor model with tumor cell lines. Inorder to establish the xenograft tumor model, 5×10⁶ viable IGROV-1 orSKBR-3 cells are injected s.c into three age and weight matched femalenude athymic mice with Matrigel (Becton Dickinson). The estimated weightof the tumor is calculated by the formula: length×(width)²/2 not toexceed 3 grams. For in vivo passaging of cells for expansion,anchorage-dependent tumor is isolated and the cells dissociated byadding 1 μg of collagenase (Sigma) per gram of tumor at 37 C overnight.

Injection of IGROV-1 cells subcutaneously gives rise to fast growingtumors while the intraperitoneal route induces a peritonealcarcinomatosis which kills the mice in 2 months. Since the IGROV-1 cellsform tumors within 5 weeks, at day 1 after tumor cell injection,monocytes as effectors are co-injected i.p. along with therapeuticantibodies ch4D5 and ch2B6 at 4 μg each per gm of mouse body weight(mbw) (Table 5). The initial injection is followed by weekly injectionsof antibodies for 4-6 weeks thereafter. Human effectors cells arereplenished once in two weeks. A group of mice will receive notherapeutic antibody but will be injected with ch4D5 N297A and humanIgG1 as isotype control antibodies for the anti-tumor and ch2B6antibody, respectively.

TABLE 5 Exemplary Experimental Set Up In Mice ch4D5 ch2B6 Human ch4D5(N297A at (N297A at (IgG1 4 μg/gm 8 mice Tumor (4 μg/gm 4 μg/gm of 4μg/gm of per cell s.c Monocytes of mbw mbw day 1 of mbw mbw day 1 groupday 0 i.p at day 1 day 1 i.p.) i.p.) day 1 i.p.) i.p.) A + − − − − −B + + − − − − C + + + − − − D + + + − + − E + + − − + − F + + − + − +

As shown in Table 5, 6 groups of 8 mice each are required for testingthe role of an anti-FcγRIIB antibody in tumor clearance with one targetand effector combination, with two different combinations of theantibody concentrations. These groups are (A) tumor cells, (B) tumorcells and monocytes, (C) tumor cells, monocytes, anti-tumor antibody,ch4D5, (D) tumor cells, monocytes, anti-tumor antibody ch4D5, and ananti-FcγRIIB antibody, e.g., ch2B6, (E) tumor cells, monocytes, and ananti-FcγRIIB antibody, e.g., ch2B6, and (F) tumor cells, monocytes,ch4D5 N297A, and human IgG1. Various combination of antibodyconcentration can be tested in similar schemes.

Studies using the breast cancer cell line, SKBR-3, are carried out inparallel with the IGROV-1 model as SKBR-3 cells over-express Her2/neu.This will increase the stringency of the evaluation of the role ofanti-FcγRIIB antibody in tumor clearance. Based on the outcome of thetumor clearance studies with the IGROV-1 cells, modifications are madeto experimental design of future experiments with other targets.

The endpoint of the xenograft tumor model is determined based on thesize of the tumors (weight of mice), survival time, and histology reportfor each group in Table 5. Mice are monitored three times a week;criteria for growing tumors are abdominal distention and presence ofpalpable masses in the peritoneal cavity. Estimates of tumor weightversus days after inoculation is calculated. Based on these threecriteria from group D mice in Table 6 versus the other groups of micewill define the role of anti-FcγRIIB antibodies in enhancement of tumorclearance. Mice that show signs of visible pain or reach 5 grams oftumor weight are euthanized with carbon dioxide and autopsied. Theantibody-treated animals are followed for two months after thistime-point.

Example 7 In Vivo Activity of FcγRIIB Antibodies in Xenograft MurineModel with Human Primary Ovarian and Breast Carcinoma Derived Cells

Primary tumors are established from primary ovarian and breast cancersby transferring tumors cells isolated from exudates from patients withcarcinomatosis. In order to translate these studies into the clinic, thexenograft model are evaluated with ascites- and pleural effusion-derivedtumor cells from two ovarian and two breast carcinoma patients,respectively. Pleural effusion, as a source of breast cancer cells, andimplantation of malignant breast tissue have been used to establishxenograft murine models successfully, see, e.g., Sakakibara et al.(1996) “Growth And Metastasis Of Surgical Specimens Of Human BreastCarcinomas In Scid Mice,” Cancer J. Sci. Am. 2: 291-300, which isincorporated herein by reference in its entirety. These studies willdetermine the broad range application of the anti-FcγRIIB antibody intumor clearance of primary cells. Tumor clearance is tested usinganti-tumor antibody, ch4D5 and anti-FcγRIIB antibody, e.g., ch2B6, inBalb/c nude mouse model with adoptively transferred human monocytes

Human ascites and pleural effusion-derived primary tumor cells. Ascitesfrom patients with ovarian cancer and pleural effusions from breastcancer patients are provided by the St. Agnes Cancer Center, Baltimore,Md. The ascites and pleural effusion from patients may contain 40-50%tumor cells and samples with a high expression of Her2neu+tumor cellswill be used to establish the xenograft models.

Ascites and pleural effusion samples are tested for expression ofHer2/neu on neoplastic cells prior to establishment of the xenografttumor model. The percentage of the neoplastic cells versus othercellular subsets that may influence the establishment of the tumor modelwill be determined. Ascites and pleural effusion from patients withovarian and breast cancer, respectively are routinely analyzed todetermine the level of expression of Her2/neu+ on the neoplastic cells.FACS analysis is used to determine the percentage of Her2/neu+neoplasticcells in the clinical samples. Samples with high percentage ofHer2/neu+neoplastic cells are selected for initiation of tumors inBalb/c mice.

Histochemistry and Immunochemistry. Histochemistry andimmunohistochemistry is performed on ascites and pleural effusion ofpatients with ovarian carcinoma to analyze structural characteristics ofthe neoplasia. The markers that are monitored are cytokeratin (toidentify ovarian neoplastic and mesothelial cells from inflammatory andmesenchymal cells); calretinin (to separate mesothelial from Her2/neupositive neoplastic cells); and CD45 (to separate inflammatory cellsfrom the rest of the cell population in the samples). Additional markersthat will be followed will include CD3 (T cells), CD20 (B cells), CD56(NK cells), and CD14 (monocytes).

For immunohistochemistry staining, frozen sections and paraffinizedtissues are prepared by standard techniques. The frozen as well as thede-paraffinized sections are stained in a similar staining protocol. Theendogenous peroxidase of the tissues is quenched by immersing the slidesin 3% hydrogen peroxide and washed with PBS for 5 minutes. Sections areblocked and the primary antibody ch4D5 is added in blocking serum for 30minutes followed by washing the samples with PBS three times. Thesecondary anti-human antibody conjugated with biotin is added for 30minutes and the slides are washed in PBS for 5 minutes. Avidin-Biotinperoxidase complex (Vector Labs) is added for 30 minutes followed bywashing. The color is developed by incubating the slides in freshsubstrate DAB solution and the reaction is stopped by washing in tapwater. For H& E staining, the slides are deparaffinized and thenhydrated in different alcohol concentrations. The slides are washed intap water and placed in hematoxylin for 5 minutes. Excess stain isremoved with acid-alcohol, followed by ammonia, and water. The slidesare placed in Eosin and followed by 90 to 100% alcohol washes fordehydration. Finally, the slides are placed in xylene and mounted withfixative for long-term storage. In all cases, the percentage of tumorcells is determined by Papanicolaou stain.

Histochemical Staining. Ascites from two different patients with ovariancarcinoma were stained by Hematoxylin and Eosin (H & E) and Giemsa toanalyze the presence of tumor cells and other cellular types. The resultof the histochemical staining is shown in FIG. 17.

Murine Models. Samples from ovarian carcinoma patients are processed byspinning down the ascites at 6370 g for 20 minutes at 4° C., lysing thered blood cells followed by washing the cells with PBS. Based on thepercentage of Her2/neu+ tumor cells in each sample, two samples, amedian and high expresser are selected for s.c inoculation to establishthe xenograft model to evaluate the role of anti-FcγRIIB antibody, inclearance of tumors. It has been reported that tumor cells make up40-50% of the cellular subset of unprocessed ascites, and afterpurification 10-50×10⁶ tumor cells were obtained from 2 liters ofascites (Barker et al. (2001) “An Immunomagnetic-Based Method For ThePurification Of Ovarian Cancer Cells From Patient-Derived Ascites,”Gynecol. Oncol. 82: 57-6382: 57-63). The isolated ascites cells areinjected i.p into mice to expand the cells. Approximately 10 mice willbe injected i.p and each mouse ascites further passaged into two miceeach to obtain ascites from a total of 20 mice, which is used to injecta group of 80 mice. Pleural effusion is handled in a manner similar toascites and Her2neu+ tumor cells are injected into the upper right andleft mammary pads in matrigel. After s.c inoculation of tumor cells,mice are followed for clinical and anatomical changes. As needed, micemay be necropsied to correlate total tumor burden with specific organlocalization.

Example 8 Effect of CH2B6 on Tumor Growth

Experimental design: Balb/c Nude female mice (Taconic, MD) were injectedat day 0 with 5×10⁶ Daudi cells subcutaneously. Mice (5 mice per group)also received i.p. injection of PBS (negative control), 10 μg/g ch4.4.20(anti-FITC antibody, negative control), 10 μg/g RITUXAN® (rituximab)(positive control) or 10 μg/g ch2B6 once a week starting at day 0. Micewere observed twice a week following injection and tumor size (lengthand width) was determined using a caliper. Tumor size in mg wasestimated using the formula: (length×width²)/2.

RESULTS: As shown in FIG. 20, Daudi cells form subcutaneous tumors inBalb/c nude females starting around day 21 post tumor cell injection. Atday 35, subcutaneous tumors were detected in mice receiving PBS (5 miceout of 5) or 10 μg/g ch4.4.20 (5 mice out of 5). Tumors were rarelydetected in mice receiving 10 μg/g RITUXAN® (rituximab) (1 mouse out of5) and were not detected in mice receiving 10 μg/g ch2B6 (0 mice out of5).

Accordingly, while the foregoing description and drawings representembodiments of the present invention, it will be understood that variousadditions, modifications, and substitutions may be made therein withoutdeparting from the spirit and scope of the present invention as definedin the accompanying claims. The presently disclosed embodiments aretherefore to be considered in all respects as illustrative and notrestrictive, the scope of the invention being indicated by the appendedclaims, and not limited to the foregoing description.

1. An antibody or fragment thereof that specifically binds theextracellular domain of human FcγRIIB and inhibits the Fc binding siteof said human FcγRIIB.
 2. The antibody or fragment thereof of claim 1,wherein said antibody enhances an immune response.
 3. The antibody orfragment thereof of claim 2, wherein said immune response is an increasein an antibody-dependent cellular response.
 4. A pharmaceuticalcomposition comprising: (i) a therapeutically effective amount of anantibody or fragment thereof that specifically binds the extracellulardomain of human FcγRIIB and inhibits binding to the Fc binding site ofhuman FcγRIIB; (ii) a cytotoxic antibody that specifically binds acancer antigen; and (iii) a pharmaceutically acceptable carrier.
 5. Thepharmaceutical composition of claim 4, wherein said cytotoxic antibodyis Herceptin®, Rituxan®, IC14, PANOREX™, IMC-225, VITAXIN™, Campath1H/LDP-03, LYMPHOCIDE™, or ZEVLIN™.
 6. The antibody or fragment thereofof claim 1 or 4 that inhibits crosslinking of FcγRIIB to animmunoreceptor tyrosine-based activation motif (ITAM) containingactivating receptor.
 7. The antibody or fragment thereof of claim 6,wherein said ITAM-containing activating receptor is an Fc receptor. 8.The antibody or fragment thereof of claim 6, wherein saidITAM-containing activating receptor is a B-cell receptor (BCR).
 9. Theantibody or fragment thereof of claim 6, wherein said inhibitionenhances the activity of said activating receptor.
 10. The antibody orfragment thereof of claim 6, wherein said inhibition leads to B cell,mast cell, dendritic cell, or macrophage activation.
 11. The antibody orfragment thereof of claim 7, wherein said Fc receptor is an FcεR or aFcγR.
 12. The antibody or fragment thereof of claim 11, wherein said Fcreceptor is FcεRI.
 13. The antibody or fragment thereof of claim 12,wherein an activity of said FcεRI is modulated by said inhibition ofcrosslinking.
 14. The antibody or fragment thereof of claim 13, whereinsaid FcεRI activity comprises modulation of calcium mobilization ormodulation of degranulation.
 15. The antibody or fragment thereof ofclaim 1 or 4, which comprises an Fc region comprising at least one aminoacid modification relative to a wild-type Fc region, such that themodified Fc region has an altered binding affinity to a Fc receptor. 16.The antibody or fragment thereof of claim 15, wherein the antibody orfragment thereof has an increased binding affinity to FcγRIIB orFcγRIII.
 17. The antibody or fragment thereof of claim 1 or 4, whereinsaid antibody is a monoclonal antibody.
 18. The antibody or fragmentthereof of claim 1 or 4, wherein said antibody is a human or humanizedantibody.
 19. The antibody or fragment thereof of claim 1 or 4, whereinsaid fragment is a F(ab′)₂ fragment.
 20. The antibody or fragmentthereof of claim 1 or 4, wherein said fragment is a F(ab) fragment. 21.A bispecific antibody comprising a first heavy chain-light chain pairthat specifically binds FcγRIIB with greater affinity than said heavychain-light chain pair binds FcγRIIA, and a second heavy chain-lightchain pair that specifically binds a tumor antigen.
 22. The bispecificantibody of claim 21, wherein said tumor antigen is HER2/neu.
 23. Abispecific antibody comprising a first heavy chain-light chain pair thatspecifically binds FcγRIIB with greater affinity than said heavychain-light chain pair binds FcγRIIA, and a second heavy chain-lightchain pair that specifically binds an FcεR.
 24. A method of treatingcancer in a patient in need thereof, wherein said cancer is associatedwith a cancer antigen, said method comprising administering to saidpatient a therapeutically effective amount of: (i) an antibody thatspecifically binds said cancer antigen; and (ii) a therapeuticallyeffective amount of an antibody or fragment thereof that specificallybinds the extracellular domain of human FcγRIIB and inhibits binding tothe Fc binding site of human FcγRIIB.
 25. A method of regulatingimmune-complex-mediated cell activation in a patient, said methodcomprising administering to said patient a therapeutically effectiveamount of an antibody or fragment thereof that specifically binds theextracellular domain of human FcγRIIB and inhibits binding to the Fcbinding site of human FcγRIIB.
 26. The method of claim 25, which resultsin an enhanced immune response.
 27. The method of claim 26, wherein saidenhanced immune response is an increase in an antibody-dependentcellular response.
 28. The method of claim 25, wherein said immunecomplex-mediated cell-activation is B cell activation, mast cellactivation, dendritic cell activation or macrophage activation.
 29. Amethod of breaking tolerance to an antigen in a patient, said methodcomprising administering to a patient in need thereof: (i) anantigen-antibody complex comprising said antigen; and (ii) an antibodyor fragment thereof that specifically binds the extracellular domain ofhuman FcγRIIB and inhibits binding to the Fc binding site of humanFcγRIIB, thereby breaking tolerance in said patient to said antigen. 30.The method of claim 29, wherein said antibody or fragment thereof isadministered before, concurrently with, or after administration of saidantigen-antibody complex.